Hbv treatment

ABSTRACT

This invention is directed to a RNA interference (RNAi) agent and the use of that RNAi agent to treat hepatitis B infection in individuals, as well as pharmaceutical compositions containing the RNAi agents of the invention. The RNAi agents, or constructs for expressing them are utilised to inhibit expression of at least one Hepatitis B virus (HBV) gene, where the agent comprises an effector sequence complementary to or substantially complementary to a predicted sequence transcribed from a target region. In some forms of the invention, the agent has more than one effector sequence. Multiple effectors may target the same region of an HBV gene, different (possibly overlapping) regions of the same gene and/or different HBV genes.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of U.S. patent application Ser. No.14/731,824, filed Jun. 5, 2015, which is a Continuation of U.S. patentapplication Ser. No. 13/882,124, filed May 23, 2013, (now U.S. Pat. No.9,080,174), which is the National Stage of International PatentApplication of PCT/CN2011/081386, filed Oct. 27, 2011, which claims thebenefit of Chinese Patent Application Serial Nos. 201010521962.3, filedOct. 28, 2010, 201010521972.7, filed Oct. 28, 2010, 201010521990.5,filed Oct. 28, 2010, 201010522003.3, filed Oct. 28, 2010,201010521948.3, filed Oct. 28, 2010, 201010521975.0, filed Oct. 28,2010, and 201010522005.2, filed Oct. 28, 2010, each of which is herebyincorporated by reference in its entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jun. 29, 2016, isnamed 34356US_CRF_sequencelisting.txt and is 2.94 bytes in size.

FIELD OF THE INVENTION

This invention is directed to an RNA interference (RNAi) agent and theuse of that RNAi agent to treat hepatitis B infection in individuals, aswell as pharmaceutical compositions containing the RNAi agents of theinvention.

BACKGROUND OF THE INVENTION

Hepatitis is a general term meaning ‘inflammation of the liver’ and hasa number of causes. Viral causes are among the most common, and may becaused by hepatitis A, B, C, D or E virus. Hepatitis B virus (HBV) inparticular is a serious and common infectious disease of the liver,affecting millions of people throughout the world.

HBV is a hepatotrophic DNA virus belonging to the Hepadnaviridae. Thefull-length of the viral genome is about 3.2 kb, and it has four openreading frames (ORFs) including surface antigen (the “S gene”), coreantigen (the “C gene”), DNA polymerase (the “P gene”) and a gene ofundetermined function referred to as the “X gene”.

More than 2,000 million people alive today have been infected with HBVat some time in their lives and of these about 350 million remainchronically infected and become carriers of the virus. HBV infection cancause acute and chronic type B hepatitis, and may eventually lead to thedevelopment of chronic hepatic insufficiency, cirrhosis, andhepatocellular carcinoma. In addition, HBV carriers can transmit thedisease for many years.

HBV is transmitted by percutaneous or parenteral contact with infectedbodily fluids or blood. The most common route of infection is viavertical transmission from mother to her baby, and in adults throughsexual intercourse or shared intravenous needles or ear-piercingequipment. Many cases of acute HBV infection occur however without atraceable route of infection.

Persons with chronic HBV infection (“carriers”—worldwide about 350-400million people) have a 12-300× higher risk of developing hepatocellularcarcinoma than non-carriers and globally HBV causes 60-80% of theworld's primary liver cancers. Every year about 25% of the over 4million acute clinical cases (i.e. 1 million people worldwide) die fromchronic active hepatitis, cirrhosis or HBV-induced liver cancer. As aconsequence, HBV ranks second only to tobacco as a known humancarcinogen.

Although vaccines against HBV has been widely used for several decades,the HBV prevalence rate in the population still remains high. Currenttherapies for chronic HBV infection have only limited inhibitory effectson viral gene expression and replication in the majority of chronicallyinfected patients. Lamivudine for example suppresses HBV replication incarriers, but the effect is reversible if therapy is stopped. Moreover,a major limitation of chronic Lamivudine therapy is the development ofviral resistance, which typically develops after 6 months of treatment.Resistance is usually associated with mutations in the highly conservedcatalytic region of the HBV polymerase gene.

For these reasons, there remains a need for a new therapeutic agent totreat HBV infection. This invention is directed to an RNA interference(RNAi) agent and the use of that RNAi agent to treat hepatitis Binfection in individuals.

The RNAi pathway is initiated by the enzyme Dicer, which cleavesdouble-stranded RNA (dsRNA) molecules into short fragments (commonlyreferred to as siRNAs) of −20-25 nucleotides. One of the two strands ofeach fragment, known as the guide strand or active strand, is thenincorporated into the RNA-induced silencing complex (RISC) throughbinding to a member of the argonaute protein family. After integrationinto the RISC, the guide strand base-pairs with its target mRNA and isthought to either inhibit a target by inhibiting translation (bystalling the translational machinery) and/or inducing cleavage of themRNA, thereby preventing it from being used as a translation template.

While the fragments produced by Dicer are double-stranded, only theguide strand, directs gene silencing. The other anti-guide strandreferred to more commonly as a passenger strand, carrier strand or *strand is frequently degraded during RISC activation (Gregory R,Chendrimada T, Cooch N, Shiekhattar R (2005). “Human RISC couplesmicroRNA biogenesis and posttranscriptional gene silencing”. Cell 123(4): 631-40). RISC assembly is thought to be governed by an enzyme thatselects which strand of a dsRNA Dicer product is loaded into RISC. Thisstrand is usually the one whose 5′ end is less tightly paired to itscomplement, and there also appears to be a clear bias for A, and to alesser extent U, at the 5′ position to facilitate binding to someargonaute proteins (Schwarz D S, Hutvágner G, Du T, Xu Z, Aronin N,Zamore P D (2003). “Asymmetry in the assembly of the RNAi enzymecomplex”. Cell 115 (2): Frank F, Sonenberg N, Nagar (2010) “Structuralbasis for 5′-nucleotide base-specific recognition of guide RNA by humanAGO2”. Nature. 465(7299):818-22).

Reference to any prior art in the specification is not, and should notbe taken as, an acknowledgment or any form of suggestion that this priorart forms part of the common general knowledge in Australia or any otherjurisdiction or that this prior art could reasonably be expected to beascertained, understood and regarded as relevant by a person skilled inthe art.

SUMMARY OF THE INVENTION

It has been discovered by the current inventors that unique sequenceswithin the Hepatitis B Virus (HBV) genome may be targeted to inhibit thevirus. By targeting specific regions of one or more genes, theexpression of those genes is inhibited, effectively “silencing” thegene. This presents a new opportunity to target HBV expression in cellsto treat HBV infection.

In one aspect of the invention, there is provided a DNA-directed RNAinterference (ddRNAi) agent (being an RNA molecule), and an expressioncassette or construct to express that agent in a cell (including invivo), for inhibiting expression of at least one Hepatitis B virus (HBV)gene, where the agent comprises an effector sequence (described furtherbelow) of at least 17 nucleotides in length complementary to orsubstantially complementary to a predicted sequence transcribed from atarget region, the target region being selected from the groupconsisting of any 10 or more contiguous nucleotides within a sequencefrom any one of SEQ ID NOS: 1-19. In an alternative embodiment, thetarget region is a sequence selected from the group consisting of any 10or more contiguous nucleotides within a sequence from any one of SEQ IDNOS: 20-27.

The effector sequence is directed to a target region of a target RNAsequence, wherein the target sequence is a transcript of a target gene.Thus the effector sequence is ‘directed to’ a target region by beingsufficiently complementary in sequence to a transcript from a targetgene containing the target region. An RNAi agent, such as a ddRNAiagent, having a double-stranded portion containing the effectorsequence, can therefore “inhibit expression of a target gene sequence”by virtue of the target gene sequence containing the target region.Accordingly, within a cell infected with HBV, the RNAi agent is capableof inhibiting expression of a target gene sequence because the sequenceof the effector (as ‘effector’ is defined below) is substantiallycomplementary to (at least) a region of the predicted mRNA targetsequence of the target gene. This can be illustrated with the followingshort sequence:

-   -   5′ATTGCG3′—DNA target sequence of gene    -   5′AUUGCG3′—mRNA target region/sequence from transcription of the        gene    -   3′UAACGC5′—effector sequence—which is substantially        complementary to a region of the predicted mRNA target sequence.

Typically, a target region is a region of an mRNA of a gene that isintended to be silenced or to have its expression (at the level oftranscription or translation) reduced.

The agent is designed so that it also comprises an effector complementsequence, ie a sequence that is substantially complementary to theeffector sequence such that it will tend to anneal so as to form adouble stranded RNA segment—the degree of complementarity required ismore particularly explained further below. Moreover, usually one end ofthe double stranded segment will be linked by a loop sequence so as toform a ‘hairpin’ shaped structure. This is also know as an ‘interruptedinverted repeat’ structure, as the DNA encoding such an RNA sequencecontains an inverted repeat of the region of the target gene that istranscribed to the effector sequence, interrupted by a stuffer or spacersequence encoding the loop.

In some forms of the invention, the agent has more than one effectorsequence. Multiple effectors may target the same region of an HBV gene,different (possibly overlapping) regions of the same gene and/ordifferent HBV genes. RNAi agents such as ddRNAi agents, can contain 2 or3 different effector sequences. As explained above, the ddRNAi agentcomprises an effector complement sequence for each effector sequence,thus forming effector—effector complement pairs (ie a firsteffector—first effector complement pair, a second effector—secondeffector complement pair, etc). These pairs may be, but need not be,contiguous to one another, as long as the RNAi agent can fold so as topermit each pair to anneal. Various other considerations suggest oneorder or another of the effectors and effector complements along thelength of the RNAi agent. Thus, embodiments of the invention include oneor more of the following:

-   -   ddRNAi agent comprising, in a 5′ to 3′ direction, a first        effector sequence; a second effector sequence; second effector        complement sequence; and a first effector complement sequence;    -   a ddRNAi agent comprising, in a 5′ to 3′ direction, a first        effector sequence; a second effector sequence; a third effector        sequence; a third effector complement sequence; a second        effector complement sequence; and a first effector complement        sequence;    -   a ddRNAi agent comprising, in a 5′ to 3′ direction, a first        effector; a first effector complement sequence; a second        effector sequence; and a second effector complement sequence;    -   a ddRNAi agent comprising, in a 5′ to 3′ direction, a first        effector sequence; a first effector complement sequence; a        second effector sequence; a second effector complement sequence;        a third effector sequence; and a third effector complement        sequence;    -   a ddRNAi agent comprising, in a 5′ to 3′ direction, a first        effector sequence; a second effector sequence; a sequence of 2        to 100 non-self-complementary nucleotides; a second effector        complement sequence; and a first effector complement sequence;    -   a ddRNAi agent comprising, in a 5′ to 3′ direction, a first        effector sequence; a sequence of 2 to 100 non-self-complementary        nucleotides; a first effector complement sequence; a second        effector sequence; a sequence of 2 to 100 non-self-complementary        nucleotides; and a second effector complement sequence.

As would be understood by one skilled in the art, and as illustrated inthe Figures, any particular effector sequence may be swapped in positionwith its complement in the agent. In particular forms of each of theembodiments described above, each effector sequence is at least 17nucleotides in length and comprises a nucleotide sequence selected fromthe group consisting of any 10 or more contiguous nucleotides from asequence from any one of SEQ ID NOS: 1-19 or SEQ ID NOS: 20-27. Theeffector sequences may all be the same, or may all be different, or maybe a combination, eg 2 effector sequences of at least 10 contiguousnucleotides of SEQ ID NO:1 and one effector sequence of at least 10contiguous nucleotides of SEQ ID NO: 4.

Preferably, the effector sequence is selected from the group consistingof any contiguous 11, 12, 13, 14, 15 or 16 nucleotides within any one ofSEQ ID NOS: 1-19 or SEQ ID NOS: 20-27, and most preferably 17 or morecontiguous nucleotides within any one of SEQ ID NOS: 1-19 or SEQ ID NOS:20-27. Typically, the effector complement will be the same length, orabout the same length (ie ±15% nucleotide length) as its correspondingeffector sequence.

In alternative embodiments, the dsRNA is comprised of 2 separate RNAstrands that are annealed to form a duplex. ddRNAi agents may beexpressed from a DNA expression cassette inserted into any suitablevector or ddRNAi construct. Accordingly, in aspects of the inventionthere is provided a ddRNAi expression cassettes comprising:

-   -   one or more promoter sequences    -   one or more DNA sequences, preferably being sequences that        encode for any 10 or more and preferably any 17 or more        contiguous nucleotides within a sequence from any one of SEQ ID        NOS: 1-19 or SEQ ID NOS: 20-27,    -   one or more DNA sequences that encode for one or more effector        complement sequences;    -   one or more terminator sequences        and optionally    -   one or more DNA sequences that encode for loop sequences; and    -   one or more enhancer sequences.

In some embodiments, one promoter is operably linked to multipleeffector-encoding regions such that it can drive expression of them,whereas in other embodiments, each effector-encoding region is operablylinked to its own promoter. In constructs where there are multiplepromoters, these may be all the same or different. Preferred promotersare U6 and H1.

There is also provided ddRNAi expression constructs, into which theddRNAi expression cassettes are inserted for expression. In addition,when the vector backbone of the construct is compatible with a deliverysystem, the ddRNAi expression constructs are also delivery constructs.

The invention also provides for siRNA agents that comprise a sequence ofat least 17 nucleotides in length selected from the group consisting ofany 10 or more contiguous nucleotides within a sequence from any one ofSEQ ID NOS: 1-19 or SEQ ID NOS: 20-27 and a sequence complement withwhich the sequence forms a duplex, and that are capable of inhibitingexpression of an HBV gene.

The invention also provides for methods of treatment of acute or chronicHBV infection in a subject, the reduction of HBV viral load in asubject, the reduction of the severity of symptoms associated with HBVinfection in a subject, and the reduction of the infectivity of HBV,comprising administering a therapeutically effective amount of a ddRNAiconstruct, ddRNAi agent or siRNA agent of the invention wherein theddRNAi construct, ddRNAi agent or siRNA agent inhibits expression of oneor more target sequences in a Hepatitis B virus (HBV) gene, preferablyat least the polymerase gene of HBV.

There is also provided a pharmaceutical composition comprising a ddRNAiagent, a ddRNAi expression cassette, a ddRNAi construct or a siRNA agentof the invention and a pharmaceutically acceptable carrier or diluent.

BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES

FIG. 1A-F illustrates some of the ddRNAi agent structures of theinvention.

FIG. 2 shows the distribution of the 642 siRNA clones obtained along theHBV polymerase gene, wherein the lines denote regions corresponding toindividual Entire siRNA Target (EsT) clones.

FIG. 3 is a comparison of the RNAi effectiveness of siRNA expressioncassettes (SECs) and their corresponding synthetic siRNAs on HBVpolymerase mRNA levels in order to validate the initial screeningresults obtained with SEC inhibition of HBV polymerase expression3.

FIG. 4 is the results of the HBV polymerase inhibition screen with 501siRNA sequences derived from the EsT library.

FIG. 5A is an illustration of the distribution of the top 100 mosteffective siRNA sequences (as identified in the large scale screen inFIG. 4) along the HBV polymerase gene. FIG. 5B illustrates how any givensequence can be mapped to the HBV polymerase gene. Shown are the areason which SEQ ID NOS: 1 to 3 are based.

FIG. 6 is a schematic of 5 individual expression cassettes and RNAiagents encoded by them, together with the effector sequence afterprocessing by Dicer. The expression cassettes are based on SEQ ID NO:3,9, 12, 13 and 23.

FIG. 7A is a schematic of a multiple effector sequence expressioncassette containing effector sequences each operably linked to separatepromoter and terminator sequences to express individual RNAi agents inthe form of a short hairpin RNAi (shRNAi) agent.

FIG. 7B is a schematic of a multiple effector sequence expressioncassette containing a first effector sequence operably linked to apromoter, and a third effector sequence operably linked to a terminatorsuch that a single multiple stem loop RNAi agent is expressed. Theexpression cassettes are based on SEQ ID NO:1, SEQ ID NO:4, and SEQ IDNO:6.

FIG. 8 is a schematic of a multiple effector sequence expressioncassette based on SEQ ID NOS: 1, 4 and 6, which gives rise to a single,long hairpin RNAi agent.

FIG. 9 illustrates the gene knockdown efficiency of SEQ ID NOS: 1 to 14and 20 to 27 following transfection into HepG2 2.2.15 cells. Knockdownefficiency was determined by qRT-PCR analysis of polymerase gene mRNA.siNC is a negative control, being a siRNA with no known target sequencesin HBV; Normal is the polymerase mRNA level in untransfected cells,standardised to a level of 1.

FIGS. 10A and 10B show luciferase activities (+/−SD; n=4) in cellstransfected with varying quantities of chemically synthesised siRNA23(A) or shRNA23 expression constructs (B) targeting pGL3-23 using theconditions listed in Tables 3 and 4. In FIG. 10A, siNC was used both asa negative control and to adjust total quantities of siRNAs added tocells to avoid potential artifacts due to unequal transfection; siRNAGL3 was used as a positive control, such that it is an siRNA targeted tothe luciferase gene. In FIG. 10B, pUC57 was used both as a negativecontrol and to adjust total quantities of plasmid DNAs added to cells toavoid potential artifacts due to unequal transfection. A plasmidexpressing a luciferase shRNA based on GL3 siRNA was used as a positivecontrol.

DETAILED DESCRIPTION OF THE EMBODIMENTS Definitions

As used herein, except where the context requires otherwise, the term“comprise” and variations of the term, such as “comprising”, “comprises”and “comprised”, are not intended to exclude further additives,components, integers or steps.

The term “RNA interference” or “RNAi” refers generally to a RNAdependent gene silencing process that is initiated by double strandedRNA (dsRNA) molecules in a cell's cytoplasm. The dsRNA reduces theexpression of a target nucleic acid sequence, which may be a DNA whoseRNA expression products are reduced, or an RNA, with which the dsRNAmolecule shares substantial or total homology.

By “double stranded RNA” or “dsRNA” it is meant a double stranded RNAmolecule that is capable of inhibiting expression of a target nucleicacid sequence with which it shares homology. In some embodiments thedsRNA is a hairpin or stem loop structure, with a duplex regionoptionally linked by at least 1 nucleotide, and is referred to as a“hairpin RNA” or “short hairpin RNAi agent” or “shRNA”. The duplex isformed between an effector sequence and a sequence complementary to theeffector sequence herein referred to as an “effector complement”.Typically, the effector complement will be the same length as itscorresponding effector sequence. As will be explained below, theeffector sequence is complementary to the target nucleic acid sequence.

An “effector sequence” is the nucleotide sequence that, when part of theRISC complex, binds to the HBV target nucleotide sequence, therebytargeting that sequence for destruction by the cell. It is analogous tothe “guide” strand discussed in the background section. The effectorsequence is ‘directed to’ a target region by being complementary orsubstantially complementary in sequence to the transcript from thetarget region such that an RNA agent having a double stranded portioncontaining the effector sequence inhibits expression of the target genesequence.

The “effector complement”, which is analogous to the passenger stranddiscussed in the background is of sufficient complementary to theeffector such that is anneals to the effector sequence. It is likelythat the effector complement will be of a similar sequence to the targetgene sequence, but does not necessarily have to be.

The term “RNAi agent” refers to a dsRNA sequence that elicits RNAi. Thisterm may be used interchangeably with “small interfering RNAs” (siRNAagents) and small hairpin RNA (shRNAi or hpRNAi agents).

The double stranded or duplex region of the RNAi agent is at least 17base pairs long, and usually in the range of 17 to 30 base pairs. RNAiagents can be synthesized chemically or enzymatically outside of cellsand subsequently delivered to cells or can be expressed in vivo by anappropriate vector in cells (see, e.g., U.S. Pat. No. 6,573,099, WO2004/106517 and WO99/49029, all of which are incorporated herein byreference).

The term “DNA-directed RNAi agent” or “ddRNAi agent” refers to an RNAiagent that is transcribed from a DNA expression cassette (“ddRNAiexpression cassette”). The ddRNAi agent transcribed from the expressioncassette may be transcribed as a single RNA that is capable ofself-annealing into a hairpin structure with a duplex region linked byat least 2 nucleotides, or as a single RNA with multiple shRNA domainsor as multiple transcripts each capable of folding as a single shRNA.

The ddRNAi expression cassette can be ligated into vectors referred toas ddRNAi vectors or ddRNAi constructs. The vectors may providesequences specifying transcription of the ddRNAi expression cassette invivo or in vitro. The vector may additionally serve as the deliveryvehicle for the ddRNAi expression cassette. Viral based vectors forexample will generate a ddRNAi construct that is useful for expressionof the ddRNAi expression cassette as well as being compatible with viraldelivery.

A cell has been “transformed”, “transduced” or “transfected” by anexogenous or heterologous nucleic acid or vector when such nucleic acidhas been introduced into the cell. The transforming DNA may or may notbe integrated (covalently linked) into the genome of the cell. Withrespect to eukaryotic cells, a stably transformed cell is one in whichthe transforming DNA has become integrated into a host cell chromosomeor is maintained extra-chromosomally (episomally) so that thetransforming DNA is inherited by daughter cells during cell replication.In non-replicating, differentiated cells the transforming DNA maypersist as an episome.

“Gene expression” can be a reference to either or both transcription ortranslation.

“Inhibition of expression” refers to the absence or observable decreasein the level of protein and/or mRNA product from the target gene. Theinhibition does not have to be absolute, but may be partial inhibitionsufficient for there to a detectable or observable change as a result ofthe administration of a RNAi or ddRNAi agent or siRNA agent or ddRNAiconstruct of the invention. Inhibition may be measured by determining adecrease in the level of mRNA and/or protein product from a targetnucleic acid relative to a cell lacking the ddRNAi agent or construct,and may be as little as 1%, 5% or 10%, or may be absolute ie 100%inhibition. The effects of inhibition may be determined by examinationof the outward properties ie quantitative and/or qualitative phenotypeof the cell or organism, and may also include an assessment of the viralload following administration of a ddRNAi agent or construct of theinvention.

As used herein, “a quantitative phenotypic trait” refers to a traitassociated with the molecular expression of a nucleic acid in a hostcell and may thus include the quantity of RNA molecules transcribed orreplicated, the quantity of post-transcriptionally modified RNAmolecules, the quantity of translated peptides or proteins, or theactivity of such peptides or proteins.

A reduction of phenotypic expression of a nucleic acid where thephenotype is a qualitative trait means that in the presence of the RNAiagent of the invention, the phenotypic trait switches to a differentstate when compared to a situation in which the RNAi agent is absent. Areduction of phenotypic expression of a nucleic acid may thus bemeasured as a reduction in steady state levels of (part of) that nucleicacid, a reduction in translation of (part of) that nucleic acid or areduction in the effect the presence of the transcribed RNA(s) ortranslated polypeptide(s) have on the eukaryotic cell or the organism,and will ultimately lead to altered phenotypic traits. It is clear thatthe reduction in phenotypic expression of a nucleic acid of interest maybe accompanied by or correlated to an observable change in phenotype.The assessment may be by way of biochemical techniques such as Northernhybridisation, quantitative real-time PCR assays, gene expressionassays, antibody binding, ELISA, RIA, western blotting and other assaysand techniques known in the art.

“Target nucleic acids” may be either RNA or DNA, whose transcriptionproducts are targeted, coding or non-coding sequence, endogenous orexogenous. In a preferred embodiment, the polymerase (P) gene of the DNAvirus hepatitis B virus is targeted for inhibition. Accordingly, in thisembodiment, the target nucleic acid is at least the RNA transcript ofthe polymerase gene.

An effector sequence for a target is complementary to or substantiallycomplementary to the predicted transcript of a region of the targetgene. By “substantially complementary” it is meant that the sequencesare hybridisable or annealable. Substantially complementary ispreferably about 85% complementary to a portion of the target gene. Morepreferably, it is at least 85-90% complementary, and most preferably atleast 95, 96, 97, 98 99 or 100% complementary. Substantialcomplementarity therefore includes 100% complementarity, but 100%complementarity may also be referred to throughout the specification as“complementary”, or “being complementary”.

A sequence complementary to or substantially complementary to a regionof a target gene has the degree of sequence complementarity across acontiguous target sequence. Generally, a double stranded RNA region ofthe invention may be subjected to mutagenesis to produce single orseveral nucleotide substitutions, deletions or additions.

A “therapeutic composition” or “pharmaceutical composition” or“composition for treating HBV infection” refers to a compositionincluding a ddRNAi agent, ddRNAi expression cassette, ddRNAi constructor siRNA agent.

The words “treat” or “treatment” refer to therapeutic treatment whereinthe object is to slow down (lessen) an undesired physiological change ordisorder. For purposes of this invention, beneficial or desired clinicalresults include, but are not limited to, alleviation of symptoms of HBVinfection, reduced infectivity of HBV, diminishment of extent ofdisease, stabilized (i.e., not worsening) state of disease, delay orslowing of disease progression, amelioration or palliation of thedisease state, and remission (whether partial or total), whetherdetectable or undetectable. “Treatment” can also mean prolongingsurvival as compared to expected survival if not receiving treatment.Treatment may not necessarily result in the complete clearance of HBVinfection but may reduce or minimise complications and side effects ofinfection and the progression of infection.

The phrase “therapeutically effective amount” means an amount of acompound of the present invention that (i) treats the particulardisease, condition, or disorder, (ii) attenuates, ameliorates, oreliminates one or more symptoms of the particular disease, condition, ordisorder, or (iii) delays the onset of one or more symptoms of theparticular disease, condition, or disorder described herein.

DETAILED DESCRIPTION

The current invention provides a new RNAi agent, and use of the RNAiagent for targeting HBV in infected individuals. Treatment of HBV isaimed at:

i. eliminating infectivity to prevent transmission and spread of HBVfrom one individual to another; andii. minimising the overall progression of liver disease within theinfected individual. ddRNAi agent

RNAi agents expressed from DNA based ddRNAi expression cassettes arereferred to as DNA-directed RNAi agents, or ddRNAi agents. They candirectly target the activity of genes with minimum off-target events. By“off target events” it is meant that expression of nucleic acids otherthan the target are not inhibited by the RNAi or ddRNAi agents. In thecase of HBV infection, this offers a unique opportunity to address theunmet clinical treatment needs for HBV. Accordingly, in one aspect ofthe invention, there is provided a DNA-directed RNA interference(ddRNAi) agent for inhibiting expression of one or more target sequencesin a Hepatitis B virus (HBV) gene, the ddRNAi agent comprising at least:

-   -   a first effector sequence of at least 17 nucleotides in length;        and    -   a first effector complement sequence;

wherein the first effector sequence is substantially complementary tothe predicted transcript of a region of the target gene.

Typically, the first effector sequence forms a double stranded regionwith the first effector complement sequence.

The sequences of the ddRNAi agents of the invention have sufficientcomplementarity to a region of the HBV gene in order to mediate targetspecific RNAi. By “substantially complementary” it is meant that thesequences are hybridisable or annealable, and either:

-   -   the sequence of the first effector sequence is at least about        80, 81, 82, 83, 84, 85, 86, 87, 88, 89 or 90% complementary to        at least 17 or more contiguous nucleotides of the target        sequence, more preferably at least about 90, 91, 92, 92, 94 or        95% complementary and even more preferably at least about 95,        96, 97, 98 or 99% complementary or absolutely complementary (ie        100%) to 17 or more contiguous nucleotides of the target        sequence; or    -   the effector sequence has at least 10 or more contiguous        nucleotides that are 100% complementary with the target and        preferably less than 6 nucleotides that cannot base pair with        the target sequence. The first effector sequence can therefore        have 1, 2, 3, 4 or 5 nucleotides that will not G-C/A-U base pair        with the target sequence. It is believed that this level of        difference will not negatively impact on the ability of the        ddRNAi agent to be able to inhibit expression of the target        sequence.

When the first effector sequence does have 1, 2, 3, 4 or 5 nucleotidesthat will not G-C/A-U base pair with the target sequence, it ispreferred that the differences are in the first or last 5 nucleotides ofthe first effector sequence, with only 1 or 2 nucleotide changes in thecentre portion of the effector sequence.

The ddRNAi agent may also comprise a first effector sequence consistingof 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotidesin length, wherein the effector sequence is substantially complementaryto the predicted transcript of a region of the target gene. A ddRNAiagent according to this embodiment of the invention therefore has amaximum length determined by the length and number of effectorsequence/s ie each effector sequence is not comprised within a longersequence.

As noted above, substantial complementarity is intended to mean that thesequences are hybridisable or annealable. The terms “hybridising” and“annealing” (and grammatical equivalents) are used interchangeably inthis specification in respect of nucleotide sequences and refer tonucleotide sequences that are capable of forming Watson-Crick base pairsdue to their complementarity. Preferably the substantially complementarysequences are able to hybridise under conditions of medium or highstringency:

-   -   high stringency conditions: 0.1×SSPE (or 0.1×SSC), 0.1% SDS, 65°        C.    -   medium stringency conditions: 0.2×SSPE (or 1.0×SSC), 0.1% SDS,        50° C.

Alternatively, “substantially complementary” would also be understood bythe person skilled in the art to involve non-Watson-Crick base-pairing,especially in the context of RNA sequences, such as a so-called “wobblepair” which can form between guanosine and uracil residues in RNA.“Complementary” is used herein in its usual way to indicate Watson-Crickbase pairing, and “non-complementary” is used to mean non-Watson-Crickbase pairing, even though such non-complementary sequences may formwobble pairs or other interactions. In the context of the presentinvention, reference to “non-pairing” sequences relates specifically tosequences between which Watson-Crick base pairs do not form.

The first effector sequence is at least 17 nucleotides long, preferably17 to 50 nucleotides and most preferably 17 to 30 nucleotides. It may be17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides inlength. When the first effector sequence is longer than 17 nucleotides,it is preferred that at least 17 contiguous nucleotides of the firsteffector sequence forms the double stranded region with thecomplementary strand.

The ddRNAi agents of the invention inhibit expression of HBV nucleicacid sequences. Preferably, the HBV target gene is the nucleic acidsequence that is expressed as the polymerase (P) gene. Accordingly, inone embodiment of the invention, the ddRNAi agent of the inventioninhibits expression of one or more target sequences in a Hepatitis Bvirus (HBV) polymerase gene. The HBV genome has overlapping open readingframes. As such, targeting particular sequences of the polymerase genewill also target the same sequences in the overlapping gene. The agentsof the invention therefore are capable of targeting multiple genes witha single effector sequence. In each preferred embodiment however, atleast the polymerase gene is targeted.

In particular embodiments, the first effector sequence is selected fromany 10 or more and preferably any 17 or more contiguous nucleotideswithin any one of the ddRNAi HBV polymerase effector sequences SEQ IDNOS: 1-19, or SEQ ID NOS: 20-27 listed below. For simplicity, the SEQ IDNOS: will be collectively referred to as SEQ ID NOS: 1 to 27.

TABLE 1 RNAi effector sequences HBV SEQ RNAi Target   Gene IDeffector sequence ^(a) Sites ^(b) nts Target ^(c)  1 GAUUGACGAUAAGGGAGA109-126 18 pol  2 UUGAAGUCCCAAUCUGGAU 2935-2953 19 pol  3GCCGGGCAACGGGGUAAAGGUUC 1139-1161 23 pol  4 UAUUUGCGGGAGAGGACAACAGA1335-1363 29 pol GUUAUC  5 UCCUGAUGUGAUGUUCUCCAUGU 155-177 23 pol &HBsAg  6 AAGGCCUCCGUGCGGUGGGG 3019-3038 20 pol  7 GGUAUUGUUUACACAGAAAGGC1116-1137 22 pol  8 GAUGUGUUCUUGUGGCAAG 908-926 19 pol  9GGGAAAGCCCUACGAACCACU 698-718 21 pol & HBsAg 10 GUGGAGACAGCGGGGUAGGC3128-3147 20 pol 11 GAGGACAACAGAGUUAUC 1335-1352 18 pol 12GCCCACUCCCAUAGGAAUUUUCC 631-653 23 pol & HBsAg 13 GGAUCUUGCAGAGUUUGG18-35 18 pol 14 CGUUGCCGGGCAACGGGGUA 1146-1165 20 pol 15GCAAUUUCCGUCCGAAGGUUUGG 575-597 23 pol & HBsAg 16 GUUGGAGGACAGGAGGUUGG340-359 20 pol & HBsAg 17 GUUGGAGGACAGGAGGUUGGUG 338-359 22 pol & HBsAg18 GAAGUGCACACGGUCCGGCAGA 1568-1589 22 pol &  X 19CAAGAUGCUGUACAGACUUGGC 762-783 22 pol & HBsAg 20 GGGAGAGGACAACAGAGUUAUC1335-1356 22 pol 21 CGGGAGAGGACAACAGAGUUAU 1336-1357 22 pol 22GCGGGAGAGGACAACAGAGUUA 1337-1358 22 pol 23 UGCGGGAGAGGACAACAGAGUU1338-1359 22 pol 24 UUGCGGGAGAGGACAACAGAGU 1339-1360 22 pol 25UUUGCGGGAGAGGACAACAGAG 1340-1361 22 pol 26 AUUUGCGGGAGAGGACAACAGA1341-1362 22 pol 27 UAUUUGCGGGAGAGGACAACAG 1342-1363 22 pol ^(a)Sequence of effector sequence based on target of HBV genome according toGenbank ID U95551 ^(b) Target position within HBV genome, based onsequence of U95551; effector sequences are the reverse complement ofthese positions. ^(c) ORFs targeted: pol corresponds to polymerase;HBsAg corresponds to the HBV surface antigen and X corresponds to the Xprotein

As explained in the background section, both strands of a dsRNA have thepotential to be the effector sequence. However there is evidence thatparticular features of a sequence can favour one strand to enter theRISC and the other strand to be destroyed. There is evidence that theprotein Argonaut 2 (AGO2) of the RISC complex has a preference forsequences with a 5′ A, and to a lesser extent a 5′ U. In addition RNAsequences with a higher AU content in 5′ regions seem to bepreferentially loaded into RISC complexes, due to a mechanism that“senses” thermodynamic stability across RNA duplexes and favorsincorporating sequences from the less stable end of the duplex. Thesesequence preferences are reflected in preferred embodiments, but are notessential.

For example, in one embodiment of this aspect of the invention, there isprovided a DNA-directed RNA interference (ddRNAi) agent for inhibitingexpression of one or more target sequences in a Hepatitis B virus (HBV)gene, the ddRNAi agent comprising at least:

-   -   a first effector sequence of any 10 or more contiguous        nucleotides within GAUUGACGAUAAGGGAGA (SEQ ID NO:1); and    -   a first effector complement sequence.

The first effector sequence is substantially complementary to thepredicted transcript of a region of the target gene.

Preferably the first effector sequence is at least 17 or more contiguousnucleotides within GAUUGACGAUAAGGGAGA (SEQ ID NO:1).

When the first effector sequence has 1, 2, 3, 4 or 5 nucleotidesdifferent to SEQ ID NO:1, the differences are preferably present in thefirst and/or last 5 nucleotides, and at least the centre 10 nucleotidesare 100% complementary to the predicted transcript of a region of thetarget gene.

In alternative embodiments, the ddRNAi agent comprises a first effectorsequence of any 10 or more, preferably any 17 or more, contiguousnucleotides within SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5,SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ IDNO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ IDNO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID: 19, SEQ ID NO: 20, SEQ ID NO:21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ IDNO: 26 and SEQ ID NO: 27.

In particularly preferred embodiments, the ddRNAi agent comprises afirst effector sequence of any 10 or more, preferably any 17 or more,contiguous nucleotides within sequences able to inhibit the expressionof a target gene region by at least 70%. Preferably, in this embodiment,the first effector is selected from SEQ ID NO:3, SEQ ID NO:9, SEQ IDNO:12, SEQ ID NO:13 and SEQ ID NO:23.

The first effector sequence may comprise a sequence selected from any 10or more and preferably any 17 or more contiguous nucleotides within asequence from the group consisting of SEQ ID NOS: 1-27, oralternatively, each effector sequence may be a variant of SEQ IDNOS:1-27, having 1, 2, 3, 4 or 5 nucleotide variations. In yet a furtherembodiment, each effector sequence may consist of 22 nucleotides, ofwhich 17, 18, 19, 20, 21 or all 22 nucleotides are contiguousnucleotides from a sequence selected from the group consisting of SEQ IDNOS: 1-27.

Multiple Targeting ddRNAi Agents

In a preferred embodiment of the invention, the ddRNAi agent comprisestwo or more effector sequences to enable targeting of more than onetarget sequence of the HBV genome. The multiple target sequences may bein the same region of the HBV gene. For example, a 17 to 30 nucleotideregion that has natural variation in the sequence between strains, orsingle nucleotide polymorphisms that have arisen to confer drugresistance. Alternatively, the target sequences may be in differentregions of the one target gene.

To provide greater specificity the ddRNAi agent comprises the following(in no particular order):

-   -   a first effector sequence of at least 17 nucleotides in length;    -   a second effector sequence of at least 17 nucleotides in length;    -   a first effector complement sequence; and    -   a second effector complement sequence;

The first and second effector sequences of a multiple targeting ddRNAiagent form a double stranded region with their respective effectorcomplements. Preferably, the first and second effector sequences are 17to 30 nucleotides in length. More preferably, the first and secondeffector sequence are both selected from any 10 or more and preferablyany 17 or more contiguous nucleotides within any one of the sequenceslisted in Table 1 above, or are sequences having 1, 2, 3, 4 or 5nucleotides difference from those sequences listed in Table 1.

In one embodiment, the first effector sequence is selected from any 10or more and preferably any 17 or more contiguous nucleotides within asequence from any one of the group consisting of SEQ ID NOS:1-27, andthe second effector sequence is selected from any 10 or more andpreferably any 17 or more contiguous nucleotides within a sequence fromany one of the group consisting of SEQ ID NOS: 1-27. The first andsecond effector sequence may both be the same sequence or mayalternatively be different sequences.

The first and second effector sequence may each comprise a sequenceselected from any 10 or more contiguous nucleotides within a sequencefrom the group consisting of SEQ ID NOS: 1-27, or alternatively, eacheffector sequence may also be a variant of SEQ ID NOS:1-27, having 1, 2,3, 4 or 5 nucleotide variations. In yet a further embodiment, eacheffector sequence may consist of 22 nucleotides, of which 17, 18, 19,20, 21 or all 22 nucleotides are contiguous nucleotides from a sequenceselected from the group consisting of SEQ ID NOS: 1-27. When there aretwo or more effector sequences, they may represent a combination of the3 types described above.

In particularly preferred embodiments, the first and second effectorsequence comprise any 10 or more, preferably any 17 or more, contiguousnucleotides within sequences able to inhibit the expression of a targetgene region by at least 70%. Preferably, in this embodiment, eacheffector sequence is selected from SEQ ID NO:3, SEQ ID NO:9, SEQ IDNO:12, SEQ ID NO:13 and SEQ ID NO:23.

The use of ddRNAi agents with multiple effector sequences has theadvantage of limiting the emergence of and targeting escape mutants,which is a problem of many current anti-viral therapies. One aspect ofthe present invention neutralizes emergent escape mutants with a ddRNAiagent that contains RNAi sequences based upon the genetic sequence ofthe target gene and additionally sequences of the point mutations thatarise to resist RNAi treatment.

Similarly, ddRNAi agents with multiple effector sequences have theadvantage of being able to target a range of sequences found indifferent viral genotypes or quasi-species, as well as the advantage ofthe additive or synergistic effects achieved with multiple effectorsequences as opposed to single effector sequences.

As mentioned above however, single effector sequences can achievemultiple targeting when the target sequence is common in 2 or moregenes. HBV contains a number of overlapping reading frames, such thattargeting a sequence in the overlapping regions will inhibit expressionof both of the genes that contain that sequence.

Long Hairpin Version

When the ddRNAi agent contains more than one effector sequence, and theddRNAi agent is expressed as a single strand of RNA, it will fold toform different structures depending on the order of the effectorsequences and the sequences complementary to the effector sequences. Inone embodiment, there is provided a DNA-directed RNA interference(ddRNAi) agent for inhibiting expression of one or more target sequencesin a Hepatitis B virus (HBV) gene, the ddRNAi agent comprising, in a 5′to 3′ direction, at least:

-   -   a first effector sequence of at least 17 nucleotides in length;    -   a second effector sequence of at least 17 nucleotides in length;    -   a second effector complement sequence; and    -   a first effector complement sequence

wherein each effector sequence is substantially complementary to thepredicted transcript of a region of the target gene. This will result ina ddRNAi agent with a structure as shown in FIG. 1A. See alsoWO2004/106517, incorporated herein by reference.

Alternatively, at least one effector, and preferably both effectorsequences, are 100% complementary to the predicted transcript of aregion of the target gene.

Preferably the first and second effector sequences are both selectedfrom the group consisting of any 10 or more and preferably any 17 ormore contiguous nucleotides within any one of SEQ ID NOS: 1-27. Forexample, in one embodiment, there is provided a DNA-directed RNAinterference (ddRNAi) agent for inhibiting expression of one or moretarget sequences in a Hepatitis B virus (HBV) gene, the ddRNAi agentcomprising, in a 5′ to 3′ direction, at least:

-   -   a first effector sequence of any 10 or more contiguous        nucleotides within GAUUGACGAUAAGGGAGA (SEQ ID NO:1);    -   a second effector sequence of any 10 or more contiguous        nucleotides within UUGAAGUCCCAAUCUGGAU (SEQ ID NO:2) or        GCCGGGCAACGGGGUAAAGGUUC (SEQ ID NO:3);    -   a second effector complement sequence; and    -   a first effector complement sequence.

Each effector sequence is substantially complementary to the predictedtranscript of a region of the target gene.

Alternatively, at least one effector, and preferably both effectorsequences, are 100% complementary to the predicted transcript of aregion of the target gene.

In particularly preferred embodiments, the first and second effectorsequence comprise any 10 or more, preferably any 17 or more, contiguousnucleotides within sequences able to inhibit the expression of a targetgene region by at least 70%. Preferably, in this embodiment, eacheffector sequence is selected from SEQ ID NO:3, SEQ ID NO:9, SEQ IDNO:12, SEQ ID NO:13 and SEQ ID NO:23.

In yet another embodiment, being an embodiment where the ddRNAi agenthas 3 effector sequences, there is provided a DNA-directed RNAinterference (ddRNAi) agent for inhibiting expression of one or moretarget sequences in a Hepatitis B virus (HBV) gene, the ddRNAi agentcomprising, in a 5′ to 3′ direction, at least:

-   -   a first effector sequence of any 10 or more contiguous        nucleotides within GAUUGACGAUAAGGGAGA (SEQ ID NO:1);    -   a second effector sequence of any 10 or more contiguous        nucleotides within UUGAAGUCCCAAUCUGGAU (SEQ ID NO:2);    -   a third effector sequence of any 10 or more contiguous        nucleotides within GCCGGGCAACGGGGUAAAGGUUC (SEQ ID NO:3);    -   a third effector complement sequence;    -   a second effector complement sequence; and    -   a first effector complement sequence.

Each effector sequence is substantially complementary to the predictedtranscript of a region of the target gene.

Alternatively, at least one effector, and optionally 2 out of the 3 orall 3 of the effectors, are 100% complementary to the predictedtranscript of a region of the target gene.

In particularly preferred embodiments, the first, second and thirdeffector sequence comprise any 10 or more, preferably any 17 or more,contiguous nucleotides within sequences able to inhibit the expressionof a target gene region by at least 70%. Preferably, in this embodiment,each effector sequence is selected from SEQ ID NO:3, SEQ ID NO:9, SEQ IDNO:12, SEQ ID NO:13 and SEQ ID NO:23.

It will also be appreciated by the skilled person that the order ofeffector and effector complements can be altered, provided that asingle, long hairpin structure is formed by annealing of the effectorsequence with its effector complement to form dsRNA. For example, in a2-effector sequence ddRNAi agent, the sequences may be arranged in thefollowing exemplary 5′ to 3′ orders:

-   -   first effector—second effector—second effector complement—first        effector complement;    -   first effector—second effector complement—second effector—first        effector complement;    -   first effector complement—second effector complement—second        effector—first effector;    -   first effector complement—second effector—second effector        complement—first effector.        In a 3-effector sequence ddRNAi agent, the sequences may be        arranged in the following exemplary 5′ to 3′ orders:    -   first effector—second effector—third effector—third effector        complement—second effector complement—first effector complement    -   first effector—second effector complement-third effector—third        effector complement—second effector—first effector complement;    -   first effector—second effector—third effector complement—third        effector—second effector complement—first effector complement    -   first effector complement—second effector complement—third        effector complement—third effector—second effector—first        effector complement    -   first effector complement—second effector complement—third        effector—third effector complement—second effector—first        effector.

In yet further embodiments, the first effector sequence may be selectedfrom any 10 or more and preferably any 17 or more contiguous nucleotideswithin a sequence from the group consisting of SEQ ID NOS:1-27; thesecond effector sequence may be selected from any 10 or more andpreferably any 17 or more contiguous nucleotides within a sequence fromthe group consisting of SEQ ID NOS:1-27; the third effector sequence maybe selected from any 10 or more and preferably any 17 or more contiguousnucleotides within a sequence from the group consisting of SEQ IDNOS:1-27; and any further effector sequences may be selected from any 10or more and preferably any 17 or more contiguous nucleotides within asequence from the group consisting of SEQ ID NOS:1-27. Alternatively,each effector sequence may also be a variant of SEQ ID NOS:1-27, having1, 2, 3, 4 or 5 nucleotide variations. Preferably, the differences arepresent in the first and/or last 5 nucleotides, and at least the centre11-12 nucleotides are 100% complementary to the predicted transcript ofa region of the target gene. Such embodiments are particularly usefulfor targeting HBV escape mutants, as well as different viral genotypesor quasi-species.

The first, second and third effector sequence may each comprise asequence selected from any 10 or more contiguous nucleotides within asequence from the group consisting of SEQ ID NOS: 1-27, oralternatively, each effector sequence may also be a variant of SEQ IDNOS:1-27, having 1, 2, 3, 4 or 5 nucleotide variations. In yet a furtherembodiment, each effector sequence may consist of 22 nucleotides, ofwhich 17, 18, 19, 20, 21 or all 22 nucleotides are contiguousnucleotides from a sequence selected from the group consisting of SEQ IDNOS: 1-27. When there are multiple effector sequences, they mayrepresent a combination of the 3 types described above.

Multiple Hairpin Version

In an alternative embodiment, there is provided a DNA-directed RNAinterference (ddRNAi) agent for inhibiting expression of one or moretarget sequences in a Hepatitis B virus (HBV) gene, the ddRNAi agentcomprising, in a 5′ to 3′ direction, at least:

-   -   a first effector sequence of at least 17 nucleotides in length;    -   a first effector complement;    -   a second effector sequence of at least 17 nucleotides in length;        and    -   a first effector complement

wherein each effector sequence is substantially complementary to thepredicted transcript of a region of the target gene.

Alternatively, at least one effector, and preferably both effectorsequences, is 100% complementary to the predicted transcript of a regionof the target gene.

This will result in a ddRNAi agent with a structure as shown in FIG. 1Bor C, depending on the type of expression cassette used to express it(see later in the specification). See also WO2005/087926 andWO2006/084209, incorporated herein by reference.

Preferably the first and second effector sequences are both selectedfrom any 10 or more and preferably any 17 or more contiguous nucleotideswithin a sequence from the group consisting of SEQ ID NOS: 1-27. Forexample, in one embodiment, there is provided a DNA-directed RNAinterference (ddRNAi) agent for inhibiting expression of one or moretarget sequences in a Hepatitis B virus (HBV) gene, the ddRNAi agentcomprising, in a 5′ to 3′ direction, at least:

-   -   a first effector sequence of any 10 or more contiguous        nucleotides within GAUUGACGAUAAGGGAGA (SEQ ID NO:1);    -   a first effector complement sequence;    -   a second effector sequence any 10 or more contiguous nucleotides        within UUGAAGUCCCAAUCUGGAU (SEQ ID NO:2) or        GCCGGGCAACGGGGUAAAGGUUC (SEQ ID NO:3); and    -   a second effector complement sequence.

Each effector sequence is substantially complementary to the predictedtranscript of a region of the target gene.

Alternatively, at least one effector, and preferably both effectorsequences, is 100% complementary to the predicted transcript of a regionof the target gene.

In particularly preferred embodiments, the first and second effectorsequence comprise any 10 or more, preferably any 17 or more, contiguousnucleotides within sequences able to inhibit the expression of a targetgene region by at least 70%. Preferably, in this embodiment, eacheffector sequence is selected from SEQ ID NO:3, SEQ ID NO:9, SEQ IDNO:12, SEQ ID NO:13 and SEQ ID NO:23.

In an embodiment where the ddRNAi agent has 3 effector sequences, thereis provided a DNA-directed RNA interference (ddRNAi) agent forinhibiting expression of one or more target sequences in a Hepatitis Bvirus (HBV) gene, the ddRNAi agent comprising, in a 5′ to 3′ direction,at least:

-   -   a first effector sequence of any 10 or more contiguous        nucleotides within GAUUGACGAUAAGGGAGA (SEQ ID NO:1);    -   a first effector complement sequence;    -   a second effector sequence of any 10 or more contiguous        nucleotides within UUGAAGUCCCAAUCUGGAU (SEQ ID NO:2);    -   a second effector complement sequence;    -   a third effector sequence of any 10 or more contiguous        nucleotides within GCCGGGCAACGGGGUAAAGGUUC (SEQ ID NO:3); and    -   a third effector complement sequence.

Each effector sequence is substantially complementary to the predictedtranscript of a region of the target gene.

Alternatively, at least one effector, and optionally 2 out of the 3 orall 3 of the effectors, is 100% complementary to the predictedtranscript of a region of the target gene.

In particularly preferred embodiments, the first, second and thirdeffector sequence comprise any 10 or more, preferably any 17 or more,contiguous nucleotides within sequences able to inhibit the expressionof a target gene region by at least 70%. Preferably, in this embodiment,each effector sequence is selected from SEQ ID NO:3, SEQ ID NO:9, SEQ IDNO:12, SEQ ID NO:13 and SEQ ID NO:23.

In yet further embodiments, the first effector sequence may be any 10 ormore contiguous nucleotides within a sequence selected from the groupconsisting of SEQ ID NOS:1-27; the second effector sequence may be any10 or more contiguous nucleotides within a sequence selected from thegroup consisting of SEQ ID NOS:1-27; the third effector sequence may beany 10 or more contiguous nucleotides within a sequence selected fromthe group consisting SEQ ID NOS:1-27; and any further effector sequencesmay be any 10 or more contiguous nucleotides within a sequence selectedfrom the group consisting of SEQ ID NOS:1-27. Preferably, each effectorsequence is at least 17 contiguous nucleotides.

Each effector sequence may also be a variant of SEQ ID NOS:1-27, having1, 2, 3, 4 or 5 nucleotide variations. Preferably, the differences arepresent in the first and/or last 5 nucleotides, and at least the centre10-12 nucleotides are 100% complementary to the predicted transcript ofa region of the target gene. Such embodiments are particularly usefulfor targeting HBV escape mutants, as well as different viral genotypesor quasi species.

The first, second and third effector sequence may each comprise asequence selected from any 10 or more contiguous nucleotides within asequence from the group consisting of SEQ ID NOS: 1-27, oralternatively, each effector sequence may also be a variant of SEQ IDNOS:1-27, having 1, 2, 3, 4 or 5 nucleotide variations. In yet a furtherembodiment, each effector sequence may consist of 22 nucleotides, ofwhich 17, 18, 19, 20, 21 or all 22 nucleotides are contiguousnucleotides from a sequence selected from the group consisting of SEQ IDNOS: 1-27. When there are multiple effector sequences, they mayrepresent a combination of the 3 types described above. Furthermore, inthe long hairpin structure or the multiple hairpin structure the ddRNAiagent may include additional effector sequences and correspondingcomplementary sequences according to one of the following formula:

Long Hairpin:

-   -   [effector sequence]₁₋₁₀ [effector complement sequence]₁₋₁₀

Multiple Hairpin

-   -   [effector sequence-effector complement sequence]₁₋₁₀

Preferably, in the long hairpin formula, the number of effectorsequences is equal to the number of effector complement sequences.Typically, there are 2, 3, 4 or 5 effector sequences, and accordingly,2, 3, 4 or 5 effector complement sequences respectively.

When the ddRNAi agent does contain more than one effector sequence, theeffector sequences may be the same or different. For example, if addRNAi agent has 3 effector sequences, 2 effector sequences may have thesame sequence, while 1 is different. Alternatively, all 3 effectorsequences may be different. Preferably, the effector sequences are any10 or more and preferably any 17 or more contiguous nucleotides within asequence selected from the group consisting of SEQ ID NOS: 1-27, orvariants of the sequences of SEQ ID NOS:1-27 which have 1, 2, 3, 4 or 5nucleotide variations. Preferably, the differences are present in thefirst and/or last 5 nucleotides, and at least the centre 10-12nucleotides are 100% complementary to the predicted transcript of aregion of the target gene.

When targeting a single region of a target sequence that has naturallyoccurring variants (eg different genotypes or quasi-species), escapemutants, or single nucleotide polymorphisms (SNPs), it is preferablethat at least one effector sequence is chosen from any 10 or more andpreferably any 17 or more contiguous nucleotides within a sequenceselected from the group consisting of SEQ ID NOS: 1-27, whereas othereffector sequences are variants of that chosen sequence. For example, afirst effector sequence may comprise 20 nucleotides of SEQ ID NO: 1; thesecond effector sequence should therefore be a variant of SEQ ID NO:1.

Hairpin Structures

In the above embodiments, the effector sequence hybridises with itscorresponding effector complement sequence to form a hairpin structure.At the end of the hairpin, two or more unbound nucleotides form the‘hinge’ or ‘loop’. In one embodiment, the unbound nucleotides are partof the effector sequence and the effector complement sequence, such thatonly a portion of the at least 17 nucleotides of the effector sequencewill form a duplex with its corresponding effector complement sequence.For example, when the effector sequence and its complement are both 22nucleotides long, 19 of the nucleotides may base pair to form a doublestranded region, leaving a total of 6 nucleotides (3 from each strand)to form a single stranded loop between and joining the effector sequenceand its effector complement sequence.

In an alternative embodiment, an additional sequence that isnon-complementary to itself, the target sequence, the effector sequenceor the sequence complementary to the effector sequence may be includedin the ddRNAi. As such, in yet another embodiment of the invention, theddRNAi agent further includes a sequence of 2 to 100 unpairednucleotides capable of forming a loop, more preferably, 2 to 10 unpairednucleotides. In a preferred embodiment the loop includes the nucleotidesequence AA, UU, UUA, UUAG, UUACAA, CAAGAGA or N1AAN2, where N1 and N2are any of C, G, U and A and may be the same or different.

There may be one or more loops depending on the ddRNAi agent structure.When a ddRNAi agent has a long hairpin structure based on formula[effector sequence] 1-10 [effector complement sequence]1-10 additionalnon-self-complementary sequence to give rise to a single loop structureis contained between the last effector sequence and the effectorcomplement sequence of that last effector sequence, as illustrated inFIG. 1D. In this embodiment, there is therefore provided a DNA-directedRNA interference (ddRNAi) agent for inhibiting expression of one or moretarget sequences in a Hepatitis B virus (HBV) gene, the ddRNAi agentcomprising, in a 5′ to 3′ direction, at least:

-   -   a first effector sequence of at least 17 nucleotides in length;    -   a second effector sequence of at least 17 nucleotides in length;    -   a sequence of 2 to 100 non-self-complementary nucleotides;    -   a second effector complement sequence; and    -   a first effector complement sequence

wherein each effector sequence is substantially complementary to thepredicted transcript of a region of the target gene.

When the ddRNAi agent has a multiple hairpin structure based on formula[effector sequence-effector complement sequence] 1-10 additionalnon-self-complementary sequence is contained between each effectorsequence and its complementary sequence to give rise to a loopstructure, as illustrated in FIGS. 1E and F (depending on the type ofexpression cassette used to express it (see later in the specification).In this embodiment, there is provided a DNA-directed RNA interference(ddRNAi) agent for inhibiting expression of one or more target sequencesin a Hepatitis B virus (HBV) gene, the ddRNAi agent comprising, in a 5′to 3′ direction, at least:

-   -   a first effector sequence of at least 17 nucleotides in length;    -   a sequence of 2 to 100 non-self-complementary nucleotides;    -   a first effector complement sequence;    -   a second effector sequence of at least 17 nucleotides in length;    -   a sequence of 2 to 100 non-self-complementary nucleotides; and    -   a second effector complement sequence

wherein each effector sequence is substantially complementary to thepredicted transcript of a region of the target gene.

In this embodiment where there are more than two effector and twoeffector complement sequences, and therefore more than two hairpinstructures, the length of additional non-self-complementary sequencethat forms each loop structure does not have to be the same. Forexample, one loop structure may have 5 nucleotides, while another loopstructure may have 9 nucleotides.

2 Strand ddRNAi Agents

As will be appreciated by one skilled in the art, it is not necessarythat the entire ddRNAi agent is expressed as one sequence. For example,in one embodiment of the invention, the first effector sequence may begenerated (e.g., transcribed by one DNA sequence), and the firsteffector complement sequence may be generated (e.g., transcribed from aseparate DNA sequence). Optionally, a loop sequence may be attached toeither transcript or part of the loop attached to the 3′ end of onetranscript and the 5′ end of the other transcript. Within the cell, thetwo transcripts then form the ddRNAi agent by hybridising throughannealing between the effector sequence/s and their complement/s.

In Vitro Expressed ddRNAi Agents or Chemically Synthesised siRNA Agents

While it is envisaged that effective treatment of chronic HBV infectionwill require ddRNAi agents to be expressed in vivo from ddRNAiconstructs (as will be outlined below), there may be circumstances whereit is desirable to administer ddRNAi agents that are expressed in vitroor to administer siRNAs that are chemically synthesised, therebyfunctioning as more of a transient therapy. Acute HBV infection forexample may benefit from a short term treatment with siRNAs that do notintegrate and replicate in the cells.

The ddRNAi agents of the invention may therefore be expressed in vitroand then delivered to target cells. Alternatively, siRNAs may bechemically synthesised and then delivered to the target cells. In lightof this, in another aspect of the invention, there is provided a smallinterfering RNAi agent (siRNA agent) for inhibiting expression of one ormore target sequences in a Hepatitis B virus (HBV) gene, the siRNAcomprising

-   -   a first effector sequence of at least 17 nucleotides in length;        and    -   a first effector complement sequence;

wherein the effector sequence is substantially complementary to thepredicted transcript of a region of the target gene.

Similarly to the ddRNAi agents described above, the siRNA agent may alsoinclude more than one effector sequence for multiple targeting. Theeffector sequences preferably target the HBV polymerase gene, and mostpreferably, are selected from any 10 or more and preferably any 17 ormore contiguous nucleotides within a sequence from the group consistingof SEQ ID NOS: 1-27.

Considerable flexibility is possible in the design of siRNAs. TypicallysiRNAs consist of dsRNA molecules with 5′-phosphate and 3′-hydroxylresidues, strand lengths can vary from 21-29 nucleotides and mayoptionally be designed to include 2 nucleotide 3′ overhangs. In someembodiments each strand can be synthesised as N19-27TT (where TT can bedeoxyribonucleotides). siRNAs can be readily designed based on regionsof SEQ ID NOS: 1-27 as described above and can be used therapeuticallyas single sequences or in any combinations. Alternatively siRNA agentscan consist of single RNA molecules containing effector and effectorcomplement sequences similar or identical to those expressed from ddRNAiexpression cassettes. These sequences can be based on SEQ ID NOS: 1-27and can be used therapeutically as single sequences or in anycombination. The siRNAs can be chemically synthesized with appropriatelyprotected ribonucleoside phosphoramidites and a conventional synthesizerand thus are widely available commercially and able to be designed andsynthesised according to routine methods in the art. In preferredembodiments, the siRNAs have the sequences of any 10 or more contiguousnucleotides within a sequence from one or more of SEQ ID NOS:1-27.

A number of transfection reagents have been used for delivering siRNAinto different cell lines. Lipofectamine 2000 and Oligofectamine areroutinely used for siRNA delivery. Naked siRNAs have also been deliveredby hydrodynamic transfection methods. Other delivery methods would beknown by the skilled person.

ddRNAi Agent Expression Cassettes

As explained above, ddRNAi agents are expressed from DNA expressioncassettes inserted into any suitable vector or ddRNAi construct. ddRNAiexpression cassettes comprise (in no particular order):

-   -   one or more promoter sequences    -   one or more DNA sequences that encode for one or more effector        sequences    -   one or more DNA sequences that encode for one or more effector        complement sequences;    -   one or more terminator sequences and optionally    -   one or more DNA sequences that encode for loop sequences    -   one or more enhancer sequences.

In one embodiment, there is provided a DNA-directed RNA interference(ddRNAi) expression cassette for expressing a ddRNAi agent, wherein theddRNAi agent inhibits expression of one or more target sequences in aHepatitis B virus (HBV) gene, the ddRNAi expression cassette comprising,in a 5′ to 3′ direction:

-   -   a promoter sequence    -   a DNA sequence that encodes for a first effector sequence    -   optionally a sequence that encodes for sequence capable of        forming a loop    -   a DNA sequence that encodes for a first effector complement        sequence; and    -   a terminator sequence.

The DNA sequence that encodes for the first effector sequence ispreferably a DNA that encodes for 10 or more, preferably 17 or more,contiguous nucleotides within a sequence from any one of SEQ ID NOS:1-27. In particularly a preferred embodiment, the first effectorsequence comprise any 10 or more, preferably any 17 or more, contiguousnucleotides within sequences able to inhibit the expression of a targetgene region by at least 70%. Preferably, in this embodiment, the firsteffector sequence is selected from SEQ ID NO:3, SEQ ID NO:9, SEQ IDNO:12, SEQ ID NO:13 and SEQ ID NO:23.

Alternatively, as outlined above in relation to the ddRNAi agent itself,the sequence that encodes for the effector sequence may encode aneffector sequence that varies by 1, 2, 3, 4 or 5 nucleotides from SEQ IDNOS:1-27 without affecting the ability of the effector sequence encodedto base pair with the target sequence and inhibit expression of thetarget sequence.

The skilled person would appreciate that a DNA sequence encoding anygiven RNA sequence is the same sequence as the RNA but having thymine(T) bases instead of uracil (U) bases.

The ddRNAi expression cassettes encoding ddRNAi agents having more thanone effector sequence in a long hairpin structure comprise, in a 5′ to3′ direction:

-   -   a promoter sequence;    -   a DNA sequence that encodes for a first effector sequence;    -   a DNA sequence that encodes for a second effector sequence;

optionally a sequence that encodes for sequence capable of forming aloop;

-   -   a DNA sequence that encodes for a second effector complement        sequence;    -   a DNA sequence that encodes for a first effector complement        sequence; and    -   a terminator sequence.

Preferably the DNA sequences encode first and second effector sequenceselected from any 10 or more and preferably any 17 or more contiguousnucleotides within a sequence from the group consisting of SEQ ID NOS:1-27. Preferably, the first and second effector sequence is selectedfrom SEQ ID NO:3, SEQ ID NO:9, SEQ ID NO:12, SEQ ID NO:13 and SEQ IDNO:23. Alternatively, the DNA sequences encode for an effector sequencethat varies from SEQ ID NOS: 1-27 by 1, 2, 3, 4 or 5 nucleotides withoutaffecting the ability of the effector sequence encoded to base pair withthe target sequence and inhibit expression of the target sequence.

When the ddRNAi agent has more than one effector sequence and a multiplehairpin structure based on formula [effector sequence-effectorcomplement sequence]1-10 expression of each [effector sequence-effectorcomplement sequence] pair may be controlled by a single promoter, oralternatively by a separate promoter. When separate promoters arecontemplated, the ddRNAi expression cassette comprises, in a 5′ to 3′direction:

-   -   a promoter sequence    -   a DNA sequence that encodes for a first effector sequence    -   a DNA sequence that encodes for a first effector complement        sequence;    -   optionally a terminator sequence;    -   a promoter sequence;    -   a DNA sequence that encodes for a second effector sequence;    -   a DNA sequence that encodes for a second effector complement        sequence; and    -   a terminator sequence.

When a single promoter is contemplated, the ddRNAi expression cassettecomprises, in a 5′ to 3′ direction:

-   -   a promoter sequence    -   a DNA sequence that encodes for a first effector sequence    -   a DNA sequence that encodes for an effector complement sequence        to the first effector sequence;    -   a DNA sequence that encodes for a second effector sequence;    -   a DNA sequence that encodes for an effector complement sequence        to the second effector sequence; and    -   a terminator sequence.

Similarly to the above embodiments, the DNA sequences preferably encodefirst and second effector sequence selected from any 10 or more andpreferably any 17 or more contiguous nucleotides within a sequence fromthe group consisting of SEQ ID NOS:1-27, or. effector sequences thatvary in sequence from SEQ ID NOS: 1-27 by 1, 2, 3, 4 or 5 nucleotides.Preferably, the first and second effector sequence is selected from SEQID NO:3, SEQ ID NO:9, SEQ ID NO:12, SEQ ID NO:13 and SEQ ID NO:23.

The ddRNAi expression cassette may alternatively be described byreference to the total length of the ddRNAi agent expressed, which is aproduct of the total length of sequence between the promoter andterminator. For example, when the length of the effector sequence in asingle effector ddRNAi consists of 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, or 30 nucleotides, the ddRNAi expression cassette willhave a length of 34 to 60 nucleotides between the promoter andterminator. This length may further include 2 to 100 nucleotides of“loop” or “hinge” sequence, giving a length of between 36 to 160nucleotides. For ddRNAi agents having multiple effector sequences, whereeach effector sequence consists of 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, or 30 nucleotides, the overall length is increasedproportionally.

One useful way of designing ddRNAi expression cassettes of the inventionis to assume Dicer cuts every 22 nucleotides (also referred to as 22 ntphasing), and processes from the base of the shRNA. The DNA sequencesthat encode effector sequences can therefore be designed to encode any10 or more, and preferably any 17 or more contiguous nucleotides withina sequence from the group consisting of SEQ ID NOS:1-27, together withappropriate spacers and other sequence requirements for the appropriatepromoter. For example, some of the sequences listed in SEQ ID NOS: 1-27are greater than 22 nucleotides; in these instances only a portion ofthe sequence needs to be operably linked to a promoter.

Agents targeting different sites of mRNA are suitable for shRNAconstruction, because they can avoid the influence of secondarystructures of mRNA, and thus perform their functions independently.

When a U6 promoter is used, it is preferable that the DNA sequenceoperably linked to the promoter starts with a guanine (G) base; when aH1 promoter is used, it is preferable that the DNA sequence operablylinked to the promoter starts with an adenine (A) base. The effectorencoding sequence can therefore be modified accordingly. For somesequences from SEQ ID NOS: 1-27, effector sequences are shorter than 22nts; in these instances it is preferable to include HBV sequencesadjacent to the HBV target sites. This would serve to maximise homologyof effector sequences to HBV mRNAs and might also permit the inclusionof A or G residues to allow efficient transcriptional initiation fromthe U6 or H1 promoters as described above. Moreover, for UStranscription in particular, the effector sequence is desirably placed3′ of the loop to avoid the 5′ G necessary for efficient U6transcription.

In some instances it may be desirable to avoid the DNA sequence TTTTwithin effector, effector complement or loop sequences since these canact as transcriptional terminators in expression constructs which usePol III promoters such as U6 or H1. shRNA design should also take in toaccount that U6 termination is expected to add a UU to the 3′ end to theshRNA. When designing long hairpin RNAs, it is sometimes advantageous tomodify the precise choice of effector sequences (either using sequencesfrom, or adjacent to SEQ ID NOS: 1-27) to maximise the likelihood thatDicer processed effector sequences will include a 5′U or A, therebyencouraging incorporation into AGO2.

The choice of whether to control expression of each [effectorsequence-effector complement sequence] pair depends on a number offactors. A single promoter may be utilised to minimise interferencebetween promoters. A ddRNAi construct with only a single promoter isalso smaller in size, which can be important in some cases for thestability of the construct, both during production (eg replication in E.coli) and delivery. In addition, the use of a single promoter avoids thepossibility of any homologous recombination between promoters.

In circumstances where a degree of regulation of expression of eacheffector sequence or complement is required though, it is advantageousto design a ddRNAi construct having multiple promoters, wherebyexpression of each [effector sequence—effector complement sequence] pairis controlled by a separate promoter. In circumstances where theeffector sequences are of a different sequence, the nature of thesequence may mean one sequence is expressed to higher expression levels.When it is desired to ensure more equal expression levels of eacheffector sequence, the more highly expressed effector sequence can bepaired with a weaker promoter and vice versa. Moreover, more efficientexpression may be achieved as the length of any one sequence to betranscribed is shorter. When multiple promoters are used, it ispreferable that not all of the promoters are the same to minimise therisk of any homologous recombination between them. In the case of 2promoters, each is preferably different. In the case of 3 promoters, atleast 2 and optionally all 3 are different from one another.

The DNA sequence encoding the effector sequence is operably linked tothe promoter sequence. A sequence is “operably linked” to anothernucleotide sequence when it is placed in a functional relationship withanother nucleotide sequence. For example, if a coding sequence isoperably linked to a promoter sequence, this generally means that thepromoter promotes transcription of the coding sequence. Operably linkedmeans that the DNA sequences being linked are typically contiguous and,where necessary to join two protein coding regions, contiguous and inreading frame. However, since enhancers may function when separated fromthe promoter by several kilobases and intronic sequences may be ofvariable length, some nucleotide sequences may be operably linked butnot contiguous.

A “promoter” or “promoter sequence” or “promoter element” is generally aDNA regulatory region capable of binding RNA polymerase in a cell andinitiating transcription of a polynucleotide or polypeptide codingsequence such as mRNA or any kind of RNA transcribed by any class of anyRNA polymerase. The promoter and terminator may be taken from differentgenes, but are typically matched to each other; that is, the promoterand terminator sequences or elements are taken from the same gene inwhich they occur naturally. Promoters also may or may not be modifiedusing molecular techniques, or otherwise, e.g., through mutation ofregulatory elements such as enhancers, to attain higher or lower levelsof transcription.

The term “constitutive” when made in reference to a promoter means thatthe promoter is capable of directing transcription of an operably linkednucleic acid sequence in the presence or absence of a specific stimulus(e.g., heat shock, chemicals, light, etc.). Typically, constitutivepromoters are capable of directing expression of a coding sequence insubstantially any cell and any tissue. The promoters used in theexpression cassettes to transcribe the ddRNAi agents preferably areconstitutive promoters, such as the promoters for ubiquitin, CMV,β-actin, histone H4, EF-1alfa or pgk genes whose expression iscontrolled by RNA polymerase II binding to the promoter, or promoterelements which are bound by RNA polymerase I. In other embodiments, aPol Il promoter such as CMV, SV40, hAAT, U1, β-actin or a hybrid Pol Ilpromoter is employed. In other embodiments, promoter elements bound byRNA polymerase III are used, such as the U6 promoters (U6-1, U6-8, U6-9,e.g.), H1 promoter, 7SL promoter, the human Y promoters (hY1, hY3, hY4(see Maraia, et al., Nucleic Acids Res 22(15):3045-52 (1994)) and hY5(see Maraia, et al., Nucleic Acids Res 24(18):3552-59 (1994)), the humanMRP-7-2 promoter, Adenovirus VA1 promoter, human tRNA promoters, the 5Sribosomal RNA promoters, as well as functional hybrids and combinationsof any of these promoters. Variants of these promoters may also beutilised, wherein the promoter is modified to decrease or increase itsactivity. For example, if a strong promoter causes too much expressionof the sequence operably linked to it, it can be modified to decreaseits activity.

When a U6 promoter is used, it is preferable that the DNA sequenceoperably linked to the promoter starts with a guanine (G) base; when aH1 promoter is used, it is preferable that the DNA sequence operablylinked to the promoter starts with an adenine (A) base. The sequences ofthe nucleic acids may therefore favour the use of one promoter overanother.

Alternatively in some embodiments it may be optimal to select promotersthat allow for inducible expression of the multiple ddRNAi agentsexpressed from the ddRNAi construct. A number of systems for inducibleexpression using such promoters are known in the art, including but notlimited to the tetracycline responsive system and the lacoperator-repressor system (see WO 03/022052 A1 Publication; and U.S.Patent Publication 2002/0162126 A1), the ecdysone regulated system, orpromoters regulated by glucocorticoids, progestins, estrogen, RU-486,steroids, thyroid hormones, cyclic AMP, cytokines, the calciferol familyof regulators, or the metallothionein promoter (regulated by inorganicmetals).

Promoters useful in some embodiments of the present invention may betissue-specific or cell-specific. The term “tissue-specific” as itapplies to a promoter refers to a promoter that is capable of directingselective expression of a nucleotide sequence of interest to a specifictype of tissue in the relative absence of expression of the samenucleotide sequence of interest in a different type of tissue (e.g.,brain). The term “cell-specific” as applied to a promoter refers to apromoter which is capable of directing selective expression of anucleotide sequence of interest in a specific type of cell in therelative absence of expression of the same nucleotide sequence ofinterest in a different type of cell within the same tissue. The term“cell-specific” when applied to a promoter also means a promoter capableof promoting selective expression of a nucleotide sequence of interestin a region within a single tissue. Alternatively, promoters may beconstitutive or regulatable. Additionally, promoters may be modified soas to possess different specificities.

In the case of HBV infection, liver specific promoters may be utilised.Examples of liver specific promoters are human Alpha Antitrypsinpromoter (hAAT) apolipoprotein H (ApoH) and Lecithin Cholesterol AcetylTransferase promoter (LCAT). Alternatively, the transthyretin or TTRpromoter may be utilised. The TTR promoter is derived from the mouseprealbumin gene and is also referred to as prealbumin. The full lengthTTR promoter normally controls expression of the serum thyroxine-bindingprotein, which is made by hepatocytes and by the choroid plexusepithelium in adults. In one embodiment, a preferred TTR promoter is aTTR promoter in which the region which controls choroids plexusexpression is deleted, but retains the regions which driveliver-specific expression. See for example WO2007/120533 andUS20060189561, incorporated herein by reference.

As noted above, enhancer elements are optionally included in the ddRNAiconstructs of the invention. One preferred enhancer element is ApoE. TheApoE enhancer element consists of approximately 155 bp derived fromapolipoprotein E (ApoE). ApoE mediates binding, internalization andcatabolism of lipoprotein particles and is a ligand for the low-densitylipoprotein (ApoB/E) receptor and for the ApoE receptor of hepatictissues. The genetic enhancer associated with the ApoE gene is aeukaryotic control element that can increase transcription of a nucleicacid specifically in the liver. The ApoE enhancer may be located up to2000 nucleotides upstream or downstream of a liver specific promoter,and may be present in more than one copy. An ApoE/hAAT is one preferredenhancer/promoter combination.

Alternatively, a synthetic enhancer (SynEnh) may be used, such as thatdescribed in US20060189561 (incorporated herein by reference).

When the ddRNAi expression cassette or construct contains more than oneterminator sequence or element, the terminator sequences or elements maybe the same, or different, or there may be a combination of terminationelements represented only once and termination elements represented twotimes or more within any cassette. Whatever terminator sequences orelements are used they should be selected to ensure that they workappropriately with the liver-specific promoter used. In instances wherePol I, Pol III or Pol III promoters are used, appropriate terminatorsequences should be employed. Termination elements useful in the presentinvention include the U1 termination sequence (U1 box), the syntheticpolyA terminator, and the so called minimal PolyA terminator.Transcriptional pause sites, such as MAZ1 and MAZ2, (See Ashfield et alEMBO J 1994 Vol 13 No 23 5656 pp and Yonaha and Proudfoot EMBO J. 2000Jul. 17; 19(14):3770-7) may be inserted upstream of the polyAterminators to assist in coupling of transcription termination andpolyadenylation. For Pol III promoters, the sequences TTTT, TTTTT orTTTTTT are commonly used as terminators. In these instances transcriptsare typically terminated by the sequence UU.

Delivery of the ddRNAi Constructs—Viral Based ddRNAi Constructs

A challenge in developing any HBV therapeutic is that virtually allhepatocytes in the patients are infected. As such, a means of achievingefficient and uniform transduction of all liver cells with the ddRNAiagent of the invention is required to provide an effective gene therapyfor chronic HBV infection. Moreover, for effective in vivo treatment ofHBV infection, the ddRNAi expression cassette of the invention has to beable to be transfected into primary cells, stem cells, and non-dividingcells.

To overcome this limitation, the ddRNAi expression cassettes of theinvention are introduced into a delivery vector, preferably derived fromviruses to ensure compatibility with viral delivery, to generate ddRNAiconstructs. As noted earlier, the vector backbone may serve the dualpurpose of being an expression vector as well as a delivery vector.Generation of the construct can be accomplished using any suitablegenetic engineering techniques well known in the art, including withoutlimitation, the standard techniques of PCR, oligonucleotide synthesis,DNA synthesis, restriction endonuclease digestion, ligation,transformation, plasmid purification, and DNA sequencing. The constructpreferably comprises, for example, sequences necessary to package theddRNAi construct into viral particles and/or sequences that allowintegration of the ddRNAi construct into the target cell genome. Theviral construct also may contain genes that allow for replication andpropagation of virus, though in preferred embodiments such genes will besupplied in trans. Additionally, the ddRNAi construct may contain genesor genetic sequences from the genome of any known organism incorporatedin native form or modified. For example, the preferred viral constructcomprises sequences useful for replication of the construct in bacteria.

The viral vector backbone may be selected from lentiviral, adenoviral(Adv), and adeno-associated viral (AAV) vectors. Non-integrating viralvectors may be used for transient expression in dividing cells of ddRNAiagents of the invention or for longer term stable expression innon-dividing cells. Integrating viral vectors, such as lentiviralvectors, mediate stable, long term expression in both dividing andnon-dividing cells.

Alternatively, minicircles such as those described in US20040214329 maybe used to deliver the ddRNAi expression cassettes. Minicircles providefor persistently high levels of nucleic acid transcription, and arecharacterised by being devoid of expression-silencing bacterialsequences.

AAV vectors are non-pathogenic and less immunogenic compared with otherviral vectors. The ability of AAV vectors to infect both dividing andnon-dividing cells, and to direct long-term gene expression in thesetissues makes it a useful vehicle for gene therapy. Moreover, AAV has avariety of pseudotypes with different tissue tropisms. A preferred AAVvector is the double stranded AAV pseudotype 8 (dsAAV8).

Typically, the genome of AAV contains only two genes. The “rep” genecodes for at least four separate proteins utilized in DNA replication.The “cap” gene product is spliced differentially to generate the threeproteins that comprise the capsid of the virus. When packaging thegenome into nascent virus, only the Inverted Terminal Repeats (ITRs) areobligate sequences; rep and cap can be deleted from the genome and bereplaced with heterologous sequences of choice. However, in order toproduce the proteins needed to replicate and package the AAV-basedheterologous construct into nascent virion, the rep and cap proteinsmust be provided in trans. The helper functions normally provided byco-infection with the helper virus, such as adenovirus or herpesvirus,can also be provided in trans in the form of one or more DNA expressionplasmids. Since the genome normally encodes only two genes it is notsurprising that, as a delivery vehicle, AAV is limited by a packagingcapacity of 4.5 single stranded kilobases (kb). However, although thissize restriction may limit the genes that can be delivered forreplacement gene therapies, it does not adversely affect the packagingand expression of shorter sequences such as ddRNAi vectors.

Accordingly, in another aspect of the invention, there is provided addRNAi construct comprising a viral vector into which a ddRNAiexpression cassette according to the invention is inserted. Preferablythe expression cassette encodes for multiple RNAi agents, as either longhairpin structures or multiple hairpin structures. In one embodiment,the viral vector is an AAV vector.

After generation of the viral based ddRNAi construct, the construct ispackaged into viral particles. Any method known in the art may be usedto produce infectious viral particles whose genome comprises a copy ofthe viral ddRNAi construct. One method utilizes packaging cells thatstably express in trans the viral proteins that are required for theincorporation of the viral ddRNAi construct into viral particles, aswell as other sequences necessary or preferred for a particular viraldelivery system (for example, sequences needed for replication,structural proteins and viral assembly) and either viral-derived orartificial ligands for tissue entry. Following transfection of the viralddRNAi construct into packaging cells, the packaging cells thenreplicate viral sequences, express viral proteins and package the ddRNAiexpression constructs into infectious viral particles. The packagingcell line may be any cell line that is capable of expressing viralproteins, including but not limited to 293, HeLa, A549, PerC6, D17,MDCK, BHK, bing cherry, phoenix, Cf2Th, or any other line known to ordeveloped by those skilled in the art. One packaging cell line isdescribed, for example, in U.S. Pat. No. 6,218,181.

Alternatively, a cell line that does not stably express necessary viralproteins may be co-transfected with one or more constructs to achieveefficient production of functional particles. One of the constructs isthe viral based ddRNAi construct; the other construct comprises nucleicacids encoding the proteins necessary to allow the cells to producefunctional virus as well as other helper functions.

The packaging cell line or replication and packaging construct may notexpress envelope gene products. In these embodiments, the gene encodingthe envelope gene can be provided on a separate construct that isco-transfected with the viral based ddRNAi construct. As the envelopeprotein is responsible, in part, for the host range of the viralparticles, the viruses may be pseudotyped. As described supra, a“pseudotyped” virus is a viral particle having an envelope protein thatis from a virus other than the virus from which the genome is derived.One with skill in the art can choose an appropriate pseudotype for theviral delivery system used and cell to be targeted. In addition toconferring a specific host range, a chosen pseudotype may permit thevirus to be concentrated to a very high titer. Viruses alternatively canbe pseudotyped with ecotropic envelope proteins that limit infection toa specific species (e.g., ecotropic envelopes allow infection of, e.g.,murine cells only, where amphotropic envelopes allow infection of, e.g.,both human and murine cells). In addition, genetically-modified ligandscan be used for cell-specific targeting, such as the asialoglycoproteinfor hepatocytes, or transferrin for receptor-mediated binding.

After production in a packaging cell line, the viral particlescontaining the ddRNAi expression cassettes are purified and quantified(titred). Purification strategies include density gradientcentrifugation, or, preferably, column chromatographic methods.

Methods of Treatment

Administration of ddRNAi agents, ddRNAi constructs or siRNA agents ofthe invention inhibit expression of HBV genes expressed in a cellinfected with HBV. Accordingly, in another aspect of the invention,there is provided a method of treating HBV infection in a subjectcomprising providing a therapeutically effective amount of a ddRNAiagent to a patient in need of treatment, wherein the ddRNAi agentinhibits expression of one or more target sequences in a Hepatitis Bvirus (HBV) gene, preferably the polymerase gene of HBV. The ddRNAiagent to be administered to the patient may be one or more of:

-   -   a ddRNAi agent comprising a first effector sequence; and a first        effector complement sequence; wherein the effector sequence is        substantially complementary to the predicted transcript of a        region of the target gene    -   ddRNAi agent comprising, in a 5′ to 3′ direction, a first        effector sequence; a second effector sequence; a second effector        complement sequence; and a first effector complement sequence,        wherein each effector sequence is substantially complementary to        the predicted transcript of a region of the target gene    -   a ddRNAi agent comprising, in a 5′ to 3′ direction, a first        effector sequence; a second effector sequence; a third effector        sequence; a third effector complement sequence; a second        effector complement sequence; and a first effector complement        sequence wherein each effector sequence is substantially        complementary to the predicted transcript of a region of the        target gene    -   a ddRNAi agent comprising, in a 5′ to 3′ direction, a first        effector sequence; a first effector complement sequence; a        second effector sequence; and a second effector complement        sequence wherein each effector sequence is substantially        complementary to the predicted transcript of a region of the        target gene    -   a ddRNAi agent comprising, in a 5′ to 3′ direction, a first        effector sequence; a first effector complement sequence; a        second effector sequence; a second effector complement sequence;        a third effector sequence; and a third effector complement        sequence; wherein each effector sequence is substantially        complementary to the predicted transcript of a region of the        target gene    -   a ddRNAi agent comprising, in a 5′ to 3′ direction, a first        effector sequence; a second effector sequence; a sequence of 2        to 100 non-self-complementary nucleotides; a second effector        complement sequence; and a first effector complement sequence        wherein each effector sequence is substantially complementary to        the predicted transcript of a region of the target gene    -   a ddRNAi agent comprising, in a 5′ to 3′ direction, a first        effector sequence; a sequence of 2 to 100 non-self-complementary        nucleotides; a first effector complement sequence; a second        effector sequence; a sequence of 2 to 100 non-self-complementary        nucleotides; and a second effector complement sequence wherein        each effector sequence is substantially complementary to the        predicted transcript of a region of the target gene.

As would be understood by one skilled in the art, and as illustrated inthe Figures, any particular effector sequence may be swapped in positionwith its complement in the agent. In particular forms of each of theembodiments described above, each effector sequence is at least 17nucleotides in length selected from the group consisting of any 10 ormore and preferably any 17 or more contiguous nucleotides within asequence from any one of SEQ ID NOS: 1-27. The effector sequences mayall be the same, or may all be different, or may be a combination eg 2effector sequences of at least 10 contiguous nucleotides of SEQ ID NO:1and 1 effector sequence of at least 10 contiguous nucleotides of SEQ IDNO: 4.

Preferably, the effector sequence is selected from the group consistingof any contiguous 11, 12, 13, 14, 15 or 16 nucleotides within any one ofSEQ ID NOS: 1-27, and most preferably 17 or more contiguous nucleotideswithin any one of SEQ ID NOS: 1-27. Typically, the effector complementwill be the same length, or about the same length (ie ±15% nucleotidelength) as its corresponding effector sequence.

Each of these ddRNAi agents may be administered via a ddRNAi expressioncassette in a ddRNAi construct, as described in the earlier sections ofthe specification. Multiple targeting may be achieved by delivering twoor more ddRNAi expression cassettes or constructs each capable ofexpressing a single ddRNAi agent, or alternatively, by delivering one ormore ddRNAi expression cassettes or constructs each capable ofexpressing more than one ddRNAi agent.

In alternative embodiments, each of the effector sequences may be 100%complementary to the predicted transcript of a region of the targetgene, or may only vary by 1, 2, 3, 4 or 5 nucleotides.

The method of treating HBV infection can optionally include apreliminary step of identifying an individual having HBV infectionincluding identification of the serotype of the HBV isolate.

For longer term or stable provision of the ddRNAi agents of theinvention, the ddRNAi agent is provided via a ddRNAi construct of theinvention ie in vivo expression of the ddRNAi agent from a ddRNAiexpression cassette inserted into a suitable vector delivered to thecell. The ddRNAi expression cassette comprises:

-   -   one or more promoter sequences    -   one or more DNA sequences selected from the group consisting of        sequences that encode for any 10 or more contiguous nucleotides        within a sequence from SEQ ID NOS: 1-27;    -   one or more DNA sequences that encode for one or more effector        complement sequences;    -   one or more terminator sequences        and optionally    -   one or more DNA sequences that encode for loop sequences    -   one or more enhancer sequences.

As outlined earlier in the specification, these components of the ddRNAiexpression cassette may have different 5′ to 3′ arrangements, all ofwhich are suitable for use in the methods of the invention.

In the methods of the invention, the HBV target gene is at least thepolymerase (P) gene, and potentially the surface antigen or X gene whenthe target sequence is contained within overlapping open reading frames,and the ddRNAi agent comprises ddRNAi effector sequences of any 10 ormore contiguous nucleotides within a sequence from SEQ ID NOS: 1-27listed in Table 1. Alternatively, as detailed earlier, the sequence thatencodes for the effector sequence or the sequence complementary to thefirst effector sequence may vary from SEQ ID NOS: 1-27 by 1, 2, 3, 4 or5 nucleotides without affecting the ability of the sequence encoded tobase pair with the target sequence and inhibit expression of the HBVtarget sequence.

Typically, each effector sequence forms a double stranded region withthe corresponding effector complement sequence.

In an alternative embodiment, the method of treating HBV infection in anindividual comprises the administration of a therapeutically effectiveamount of a ddRNAi construct that encodes a ddRNAi agent having morethan one effector sequence, such as those listed above.

The HBV infection to be treated may be a chronic HBV infection. By“chronic” it is meant that the infection with HBV is long-lasting orpersistent. The term chronic describes the course of the disease, or itsrate of onset and development. When treating chronic HBV infection, itis preferable that the ddRNAi construct is administered and eitherstably maintained or integrated in to the target cell to ensure longerterm expression of the ddRNAi agent. As detailed above, this can beachieved with the use of a ddRNAi construct having a viral vectorbackbone. In accordance with this, there is provided a method oftreating chronic HBV infection in an individual comprising theadministration of a therapeutically effective amount of a ddRNAiconstruct of the invention to a patient in need of treatment.

Treatment of chronic HBV infection is aimed at reducing the infectivityof the virus by interfering with viral replication. This in turn lowersthe risk of transmission and spread of HBV. There is therefore provideda method of reducing the infectivity of HBV in an individual infectedwith HBV, comprising administering to the individual a ddRNAi constructsof the invention to target a HBV gene, preferably the polymerase gene.The clinical endpoints used in chronic hepatitis B therapy will differbetween patients with compensated hepatitis B (where sustained viralsuppression, serological response, histological improvement,normalisation of liver function tests and non-clinical progression arekey endpoints) and those with decompensated hepatitis B. In patientswith decompensated disease the liver is extensively scarred and fibroticand prevention/reversal of liver damage and prevention of diseaseprogression and obviation of liver transplantation are the mainobjectives of therapy. Endpoints for compensated hepatitis B arereduction of viral load with serological and biochemical improvement,histological improvement, measure of liver failure and end stage liverdisease, complications, transplantation and mortality.

In another embodiment, there is provided a method of minimisingprogression of liver disease in a subject as a result of HBV infection,comprising administering to the individual a ddRNAi construct of theinvention to target a HBV gene, preferably the polymerase gene.

In yet another embodiment of the invention, there is provided a methodof minimising the symptoms associated with HBV infection in a subject,comprising administering to the individual a ddRNAi construct of theinvention to target a HBV gene, preferably the polymerase gene.

The status or severity of an individual's HBV infection may be assessedby determining their viral load. By “viral load” it is meant the amountof virus in an involved body fluid. For example, it can be given in RNAcopies per millilitre of blood plasma. Individuals having a high viralload are considered to have a more severe HBV infection. Alternatively,the viral load is an indicator of the responsiveness of an individual toa particular treatment. If a treatment is working and keeping the levelsof virus low, it is indicative of a successful treatment. It istherefore an objective of treatment to lower an individual's viral load.Accordingly, there is also a provided a method of lowering a viral loadof an individual with an HBV infection, comprising administering to theindividual a ddRNAi construct of the invention to target a HBV gene,preferably the polymerase gene.

Subsequent monitoring of the viral load in the individual who hasreceived treatment with a ddRNAi construct of the invention is thereforea phenotypic indicator of the effectiveness of the ddRNAi construct andthe ddRNAi agent of the invention.

Alternatively, treatment of acute HBV infection may not require longterm treatment, and it may in fact be preferred to rely on the transientpresence of a ddRNAi agent or siRNA agent as opposed to long termexpression of ddRNAi agents from integrated or stably maintained ddRNAiconstructs. By “acute” it is meant that the HBV infection has a rapidonset, and/or a short duration.

In accordance with this embodiment of the invention, there is provided amethod of treating acute HBV infection in an individual comprising theadministration of a therapeutically effective amount of an in vitrosynthesised or chemically synthesised ddRNAi agent to a patient in needof treatment, for inhibiting expression of one or more target sequencesin a Hepatitis B virus (HBV) gene, the ddRNAi agent comprising at least:

-   -   a first effector sequence of at least 17 nucleotides in length        selected from any 10 or more contiguous nucleotides within a        sequence from the group consisting of SEQ ID NOS: 1-27; and    -   a first effector complement sequence;

wherein the effector sequence is substantially complementary to thepredicted transcript of a region of the target gene. In this embodiment,the ddRNAi agent of the invention is produced in vitro or chemicallysynthesised and provided to the cell.

Any of the siRNA agents or ddRNAi agents of the invention describedthroughout the specification are suitable for in vitro expression anddelivery to the cell.

Preferably, the HBV target gene is at least the polymerase (P) gene, andpotentially the surface antigen, core antigen or X gene when the targetsequence is contained within overlapping open reading frames.Accordingly, in one embodiment of the invention, the ddRNAi agentinhibits expression of one or more target sequences in a Hepatitis Bvirus (HBV) polymerase gene. The first effector sequence is preferablyselected from any 10 or more and preferably any 17 or more contiguousnucleotides within a sequence from the effector sequences SEQ ID NOS:1-27 listed in Table 1. Alternatively, the effector sequence may varyfrom SEQ ID NOS: 1-27 by 1, 2, 3, 4 or 5 nucleotides without affectingthe ability of the effector sequence to base pair with the targetsequence and inhibit expression of the HBV polymerase gene.

In an alternative embodiment, an siRNA agent may be administered.Preferably the siRNA agents, similarly to the ddRNAi agent, targets theHBV polymerase gene, and may have a sequence selected from any 10 ormore and preferably any 17 or more contiguous nucleotides within asequence from the group consisting of SEQ ID NOS: 1-27, or may vary fromSEQ ID NOS: 1-27 by 1, 2, 3, 4 or 5 nucleotides without affecting theability of the effector sequence to base pair with the target sequenceand inhibit expression of the HBV polymerase gene.

Administration of a ddRNAi agent or siRNA agent of the invention to anindividual with an acute HBV infection can also lower a viral load,reduce the severity of symptoms associated with the acute infection, andreduce the infectivity of HBV.

In another aspect of the invention, there is provided the use of theddRNAi constructs, ddRNAi agents or siRNA agents of the invention in thepreparation of medicaments for treatment of HBV infection, preferablychronic HBV infection, the reduction of HBV viral load, the reduction ofthe severity of symptoms associated with HBV infection, and thereduction of the infectivity of HBV.

In a further aspect of the invention there is provided ddRNAiconstructs, ddRNAi agents or siRNA agents for treating HBV infection,preferably chronic HBV infection, reducing HBV viral load, reducing theseverity of symptoms associated with HBV infection, and reducing theinfectivity of HBV.

In a further aspect of the invention there is provided a compositioncomprising ddRNAi constructs, ddRNAi agents or siRNA agents as an activeingredient for treating HBV infection, preferably chronic HBV infection,reducing HBV viral load, reducing the severity of symptoms associatedwith HBV infection, and reducing the infectivity of HBV.

The one or more effector sequences of the ddRNAi constructs, ddRNAiagents or siRNA agents used in the methods of the invention comprise any10 or more, preferably any 17 or more, contiguous nucleotides withinsequences able to inhibit the expression of the HBV target gene regionby at least 70%. Preferably the one or more effector sequence isselected from SEQ ID NO:3, SEQ ID NO:9, SEQ ID NO:12, SEQ ID NO:13 andSEQ ID NO:23.

Pharmaceutical Compositions

The ddRNAi agents, the siRNA agents or the vectors comprising ddRNAiexpression cassettes of the invention can be formulated intopharmaceutical compositions by combination with appropriate,pharmaceutically acceptable carriers or diluents. Accordingly, there isprovided a pharmaceutical composition comprising a ddRNAi agent, addRNAi expression cassette, a ddRNAi construct or a siRNA agent of theinvention and a pharmaceutically acceptable carrier or diluent.

In pharmaceutical dosage forms, the agents or the vectors comprising theddRNAi expression cassettes may be administered alone or in associationor combination with other pharmaceutically active compounds. Those withskill in the art will appreciate readily that dose levels for agents orvectors comprising the ddRNAi expression cassettes will vary as afunction of the nature of the delivery vehicle, the relative ease oftransduction of the target cells, the expression level of the RNAiagents in the target cells and the like.

The ddRNAi agents, the siRNA agents or the vectors comprising ddRNAiexpression cassettes of the invention can be formulated intopreparations for injection or administration by dissolving, suspendingor emulsifying them in an aqueous or non aqueous solvent, such as oils,synthetic aliphatic acid glycerides, esters of higher aliphatic acids orpropylene glycol; and if desired, with conventional additives such assolubilisers, isotonic agents, suspending agents, emulsifying agents,stabilizers and preservatives.

Pharmaceutically acceptable carriers or diluents contemplated by theinvention include any diluents, carriers, excipients, and stabilizersthat are nontoxic to recipients at the dosages and concentrationsemployed, and include buffers such as phosphate, citrate, and otherorganic acids; antioxidants including ascorbic acid and methionine;preservatives (such as octadecyldimethylbenzyl ammonium chloride;hexamethonium chloride; benzalkonium chloride, benzethonium chloride;phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol);low molecular weight (less than about 10 residues) polypeptides;proteins, such as plasma albumin, gelatin, or immunoglobulins;hydrophilic polymers such as polyvinylpyrrolidone; amino acids such asglycine, glutamine, asparagine, histidine, arginine, or lysine;monosaccharides, disaccharides, and other carbohydrates includingglucose, mannose, or dextrins; chelating agents such as EDTA; sugarssuch as sucrose, mannitol, trehalose or sorbitol; salt-formingcounter-ions such as sodium; metal complexes (e.g. Zn-proteincomplexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ orpolyethylene glycol (PEG).

The pharmaceutical composition may be prepared for various routes andtypes of administration. In general the formulations are prepared byuniformly and intimately bringing into association the active ingredientwith liquid carriers or finely divided solid carriers or both, and ifnecessary, shaping the product. Formulation may be conducted by mixingat ambient temperature at the appropriate pH, and at the desired degreeof purity, with physiologically acceptable carriers, i.e., carriers thatare non-toxic to recipients at the dosages and concentrations employed.

The one or more effector sequences of the ddRNAi constructs, ddRNAiagents or siRNA agents used in the compositions of the inventioncomprise any 10 or more, preferably any 17 or more, contiguousnucleotides within sequences able to inhibit the expression of the HBVtarget gene region by at least 70%. Preferably the one or more effectorsequence is selected from SEQ ID NO:3, SEQ ID NO:9, SEQ ID NO:12, SEQ IDNO:13 and SEQ ID NO:23.

EXAMPLES

The following examples are illustrative in nature and are in no wayintended to be limiting.

Example 1 Production of an Entire siRNA Target (EsT) Library

The full length HBV RNA-dependent DNA polymerase gene from HBV Adr-1(Accession M38454) was sub-cloned and used to generate an Entire siRNATarget (EsT) library. 5000 clones were sequenced, of which 642 wereidentified as having non-repeat sequences ranging from 19-23 bp. Thesequences, which represent potential targets for the ddRNAi agents ofthe invention, were distributed along the target gene (FIG. 2).

Example 2 Large Scale Screening Results Using SECs with ≧50% Knock Down

To identify the most effective siRNA sequences which could then be usedto prepare the ddRNAi agents of the invention, siRNA expressioncassettes (SECs) amplified by PCR were transfected into HepG2 2.2.15cells. Effects on the levels of HBV polymerase mRNA expression wasevaluated to identify functional siRNA sequences.

40 siRNAs that corresponded to the first SECs screened were chemicallysynthesised and transfected into HepG2 2.2.15 cells to validate theoriginal polymerase mRNA knock down levels. HepG2 2.2.15 is a stablecell line containing an integrated tandem dimer of the HBV genome(Genebank Accession #: U95551), and can stably express HBV antigens andHBV Dane particles. The correlation results are shown in FIG. 3.

As can be seen from FIG. 3, when SECs inhibition of polymeraseexpression was >50%, most of the corresponding synthetic siRNA sequencesgave around 70% knockdown of HBV mRNA, and only 2/23 gave knockdownlevels below 50%. In contrast to the SECs with <50%, silencingefficiency, only 18% (3/17) of the corresponding synthetic siRNAsproduced >70% knock down (p<0.05—Pearson's chi-square test, |tStat|=4.29>1.68). Furthermore, a synthetic siRNA (need explanation ofthis) which produced >90% knock down corresponded to a SEC clone whichgave >65% knock down. From this it was concluded that a reasonablecorrelation existed between the knock down of HBV mRNA by siRNA fromSECs and that produced by synthetic siRNAs, to a cut-off value of ≧50%knock down by SECs. This cut off value was used for the rest of thescreening of the SECs.

Example 3 Target Screening by siRNA Expression Cassettes (SECs)

FIG. 4 shows the results of large scale screening in vitro forinhibition of HBV mRNA accumulation (501 non-repeated siRNA agents). Ofthose sequences screened, 100 siRNA sequences were effective in knockingdown HBV mRNA by ≧50%, 14 of which resulted in >70% knock down (Table2). The distribution of the top 100 siRNA targets on the HBV polymerasegene is shown in FIG. 5A; any sequence can in turn be mapped on thepolymerase. FIG. 5B for example maps the first 3 sequences.

TABLE 2 SEQ  Relative   ID Length mRNA NO RNAi effector sequence ^(a)(bp) expression  1 GAUUGACGAUAAGGGAGA 18 0.1  2 UUGAAGUCCCAAUCUGGAU 190.14  3 GCCGGGCAACGGGGUAAAGGUUC 23 0.19  4 UAUUUGCGGGAGAGGACAACAGAGUUAUC29 0.25  5 UCCUGAUGUGAUGUUCUCCAUGU 23 0.25  6 AAGGCCUCCGUGCGGUGGGG 200.26  7 GGUAUUGUUUACACAGAAAGGC 22 0.26  8 GAUGUGUUCUUGUGGCAAG 19 0.27  9GGGAAAGCCCUACGAACCACU 21 0.27 10 GUGGAGACAGCGGGGUAGGC 20 0.28 11GAGGACAACAGAGUUAUC 18 0.29 12 GCCCACUCCCAUAGGAAUUUUCC 23 0.29 13GGAUCUUGCAGAGUUUGG 18 0.29 14 CGUUGCCGGGCAACGGGGUA 20 0.29

Relative mRNA expression was measured (by a one-step quantitative RT-PCRmethod using SensiMix SYBR One-Step Kit (Bioline, USA) according to themanufacturer's guidelines) in transfected HepG2 2.2.15 cells relative toan empty vector control whose level was arbitrarily set at 1.0. Thespecific primers for the HBV polymerase gene were forward primer5′-TGTGGTTATCCTGCGTTAATG-3′ Reverse primer 5′-GCGTCAGCAAACACTTGG-3′, thePCR product was 158 bp long. U6 snRNA (forward primer5′-CTCGCTTCGGCAGCACA-3′ Reverse primer 5′-AACGCTTCACGAATTTGCGT-3′) wasused as the internal control, the PCR product was 94 bp long. Therelative expression level of Polymerase gene was normalized using the2-ΔΔCt analysis method. The experiments were performed usingquadruplicate independent transductions.

Example 4 Confirmation of Activity Using Synthesised siRNAs

The first round of screening used a vector with opposing promoters(pU6H1-GFP). In shRNA expressing constructs the shRNA is under thecontrol of a single promoter. Accordingly, to confirm the efficacy ofthe top 14 siRNA sequences shown in Table 2 prior to developing shRNAexpression constructs based on them, HepG2 2.2.15 cells were transfectedwith each siRNA and inhibition of HBV replication was assayed todetermine HBV polymerase mRNA levels.

The 14 siRNAs were chemically synthesized with a dTdT 3′ overhang.5′-FAM labelled siRNA-GL3 (pGL3 Luciferase Reporter Vector withunrelated siRNA sequence) served as negative control. The siRNAs weredissolved in 100 uM in DEPC-treated water, aliquoted out and stored at−20° C. In addition, SEQ ID NO:4, which is 29 bp long, was redesignedinto 8 siRNAs (SEQ ID NOS:20 to 27), each of 22 by made up ofoverlapping sequence from SEQ ID NO:4 (Table 3 below):

TABLE 3 SEQ ID NO RNAi effector sequence  4UAUUUGCGGGAGAGGACAACAGAGUUAUCTT 20 GGGAGAGGACAACAGAGUUAUCTT 21CGGGAGAGGACAACAGAGUUAUTT 22 GCGGGAGAGGACAACAGAGUUATT 23UGCGGGAGAGGACAACAGAGUUTT 24 UUGCGGGAGAGGACAACAGAGUTT 25UUUGCGGGAGAGGACAACAGAGTT 26 AUUUGCGGGAGAGGACAACAGATT 27UAUUUGCGGGAGAGGACAACAGTT

Transfection optimization was performed by varying 5′ FAM-labelledsiRNA-GL3 and Lipofectamine 2000 (Invitrogen). The cells were maintainedin DMEM (GIBCO, USA) supplemented with 10% PBS (Excellbio, China) and200 μg/mL G418 (Sangon, China) at 37° C., 5% CO2.

HepG2 2.2.15 cells were transfected with siRNAs using the optimisedprotocol and harvested 72 h after transfection. Total RNA was isolatedusing Trizol (Invitrogen) and quantified in a one-step quantitativeRT-PCR method using SensiMix SYBR One-Step Kit (Bioline, USA) accordingto the manufacturer's guidelines as already detailed in Example 3.

Results

The results are expressed as mean±STDV (Table 4 and FIG. 9).

SEQ Polymerase mRNA Level ID NO Lot 1 Lot 2 Lot 3 Lot 4 AVERAGE STDV 10.39 0.52 0.51 0.72 0.54 0.1373 2 0.79 0.24 0.18 0.43 0.41 0.2722 3 0.580.27 0.18 0.14 0.29 0.2014 4 0.41 0.34 0.22 0.33 0.0928 5 0.34 0.35 0.440.37 0.37 0.0450 6 0.36 0.32 0.26 0.54 0.37 0.1190 7 0.45 0.30 0.26 0.630.41 0.1702 8 0.48 0.46 0.33 0.72 0.50 0.1653 9 0.45 0.26 0.20 0.28 0.300.1068 10 0.17 0.40 0.43 0.67 0.42 0.2053 11 0.32 0.33 0.30 0.30 0.310.0178 12 0.32 0.28 0.16 0.26 0.25 0.0704 13 0.23 0.25 0.23 0.26 0.240.0152 14 0.36 0.34 0.38 0.36 0.36 0.0180 20 0.43 0.23 0.19 0.30 0.290.1027 21 0.33 0.40 0.24 0.19 0.29 0.0915 22 0.37 0.51 0.84 0.27 0.500.2453 23 0.18 0.29 0.20 0.32 0.25 0.0648 24 0.31 0.38 0.46 0.45 0.400.0686 25 0.50 0.33 0.54 0.25 0.41 0.1400 26 0.43 0.41 0.32 0.43 0.400.0518 27 0.38 0.44 0.33 0.31 0.37 0.0585 siNC 0.72 0.73 0.69 0.78 0.730.0398 Normal 1.00 1.00 1.00 1.00 1.00 0.0000 siNC = negative control;Normal = untransfected cellsOf the 22 siRNAs tested, all exhibited at least about 50% inhibition,and most exhibited 60 to 70% inhibition. Despite the high sequencesimilarity between the 22 bp variants SEQ ID NO: 20 to 27 of SEQ IDNO:4, the ability to inhibit expression of their sequence varied from 50to 71%. Based on these results, SEQ ID NOS: 3, 9, 12, 13 and 23 wereselected for further development. While all of those SEQ ID NOS targetthe polymerase gene, these sequences were also selected on the basis oftheir spread along the polymerase gene mRNA (see column “HBV TargetSites” in Table 1).

Example 5 shRNA Design and Construction Step 1—Effector Sequence Design

shRNA expression constructs were designed based on SEQ ID NOS: 3. 9, 12,13 and 23. Constructs can be readily synthesised using a variety ofpromoters, including different versions of human U6 promoters withdifferent intrinsic activities (Domitrovich, A M and Kunkel, GR (2003)Multiple, dispersed human U6 small nuclear RNA genes with variedtranscriptional efficiencies Nucleic Acids Research 31: 2344-2352) orvarious pol II promoters. In designing these constructs, the followingconsiderations were applied:

-   -   Dicer processes from the base of shRNAs    -   Dicer processing is imprecise but is predominantly expected to        cut every 22 nucleotides    -   U6 termination is expected to add a UU to the 3′ end to the        shRNA    -   the processed effector sequence desirably contains a 5′ U or A        to facilitate efficient Ago 2 loading.    -   the effector sequence is positioned 3′ of the loop to avoid the        5′ G preferred for maximal U6 transcription    -   5′ A or U residues can be incorporated into shRNA effector        sequences by incorporating sequences 2 or 3 nucleotides upstream        or downstream of the sequences targeted by the RNAi agent; this        is referred to as “sliding” sequences 1, 2 or 3 nucleotides ie        the effector sequence for shRNAs can be based on sequences 1, 2        or 3 nucleotides 5′ or 3′ relative to the 5′ end of the siRNA        effector sequence.

When applied to the 5 siRNAs based on SEQ ID NOS: 3. 9, 12, 13 and 23:

i) For a shRNA based on SEQ ID NO:3: gccgggcaacgggguaaagguucTT“Best” shRNA effector sequence (minimize 5′ GC sequences, slide “down” 3nts)

GGGCAACGGGGUAAAGGUUCuu (uu from pol III termination)

ii) For a shRNA based on SEQ ID NO:9: gggaaagcccuacgaaccacuTT“Best” shRNA effector sequence (minimize 5′ Gs, slide down 3 nts to 5′A, add GA to 3′ (from HBV genome to maximize homology)

AAAGCCCUACGAACCACUGAuuiii) For a shRNA based on SEQ ID NO:12: gcccacucccauaggaauuuuccTT“Best” shRNA effector sequence move up 2 nts to avoid UUUU (will causepremature termination) on antisense strand, add AG from HBV sequence,which fortuitously incorporates a 5′ A.

AGGCCCACUCCCAUAGGAAUUiv) For a shRNA based on SEQ ID NO:13: ggaucuugcagaguuuggTT“Best” shRNA effector sequence (increase length to 20 nts, slide up 2nts, add TG from HBV sequence, which results in a 5′ T (ie U)

TGGGAUCUUGCAGAGUUUGGuuv) For a shRNA based on SEQ ID NO:23: ugcgggagaggacaacagaguuTT“Best” shRNA effector sequence reduce size at 3′ end by 2 nts

UGCGGGAGAGGACAACAGAGUU

Step 2—Expression Cassette

U6 shRNA cassettes (Genscript) based on the effector sequences designedin step (a) were prepared and cloned in to pUC57.

The inserts of the expression cassettes, which included some flankingrestriction sites for subsequent manipulation, had the followingsequences:

U6.HBVshRNA 3 (SEQ ID NO: 28)GGGACCCGGTACCTCGAGAGATCTGGGCAGGAAGAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTAGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCGAACCTTTACCCCGTTGCCCGGCAAGAGACCGGGCAACGGGGTAAAGGTTCTTTTTTGTTAACGAAT TC U6.HBVshRNA 9(SEQ ID NO: 29) GGGACCCGGTACCTCGAGAGATCTGGGCAGGAAGAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTAGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCGTCAGTGGTTCGTAGGGCTTTCCCAAGAGAGGAAAGCCCTACGAACCACTGATTTTTTGTT AACGAATTCU6.HBVshRNA 12 (SEQ ID NO: 30)GGGACCCGGTACCTCGAGAGATCTGGGCAGGAAGAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTAGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCGATTCCTATGGGAGTGGGCCTCACAAGAGATGAGGCCCACTCCCATAGGAATTTTTTTGTTAACGA  ATTC U6.HBVshRNA 13(SEQ ID NO: 31) GGGACCCGGTACCTCGAGAGATCTGGGCAGGAAGAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTAGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCGCCAAACTCTGCAAGATCCCAGACAAGAGATCTGGGATCTTGCAGAGTTTGGTTTTTTG TTAACGAATTCU6.HBVshRNA 23 (SEQ ID NO: 32)GGGACCCGGTACCTCGAGAGATCTGGGCAGGAAGAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTAGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCGCTCTGTTGTCCTCTCCCGCAAACAAGAGATTTGCGGGAGAGGACAACAGAGTTTTTTGTTAACGAATTC 

The corresponding expressed shRNA for each of these expression cassettesare shown in FIG. 6 (as SEQ ID NOS: 36 to 40). Symbol “̂” and “v”indicates potential processing sites of shRNA, resulting from eithertranscriptional initiation/termination or Dicer processing with a 22 ntphase and 2 nt 3′ overhang, the predicted effector sequence assumingsuch processing is shown below.

Example 6 Generation of Stable shRNA Expressing Cells Using MultipleSequence Constructs

As outlined above, one useful way of designing ddRNAi expressioncassettes of the invention is to assume Dicer cuts every 22 nucleotides(also referred to as 22 nt phasing). The DNA sequences that encodeeffector sequences can therefore be designed to encode any 10 or more,and preferably any 17 or more contiguous nucleotides within a sequencefrom the group consisting of SEQ ID NOS:1-27, together with appropriatespacers and other sequence requirements for the promoter and/orterminator.

ddRNAi constructs will be generated with the following structures (usingnon-limiting exemplary sequences):

i) Single Hairpin Expression Cassette

As mentioned above, FIG. 6 illustrates the expression cassette,expressed hairpin RNAi agent and theoretical effector sequence processedby Dicer (assuming 22 nucleotide phasing) based on SEQ ID NO: 3 (FIG.6A), SEQ ID NO:9 (FIG. 6B) and SEQ ID NO:12 (FIG. 6C), SEQ ID NO: 13(FIG. 6D) and SEQ ID NO: 23 (FIG. 6E). In the cassette of FIGS. 6A and6B, 20 contiguous nucleotides of SEQ ID NO:3 and 9 respectively areencoded for; in the cassette of FIG. 6C to 6E, 21 contiguous nucleotidesof SEQ ID NO: 12, 13 and 23 respectively are encoded for. The arrows onthe hairpin RNAi agent indicate where Dicer is expected to cut toproduce the effector sequence shown underneath.

The sequence of the expression cassettes of FIGS. 6A to 6E have alreadybeen provided above (SEQ ID NOS: 28 to 32).

ii) Multiple Hairpin Expression Cassette

FIG. 7 illustrates the expression cassette, expressed hairpin RNAi agent(SEQ ID NOS: 41 to 43 and theoretical effector sequence processed byDicer (assuming 22 nucleotide phasing) based on SEQ ID NO: 1, SEQ IDNO:4 and SEQ ID NO:6 when arranged within the cassette to give rise toexpression of individual hairpin RNAi agents (FIG. 7A) or a single RNAcontaining the 3 hairpin RNAi agents prior to processing (FIG. 7B). SEQID NOS:1, 4 and 6 are the same as described above in relation to thesingle hairpin expression cassette. The “

” and “v” symbols on the hairpin RNAi agent indicate where Dicer isexpected to cut to produce the effector sequence shown underneath.

The expression cassette of FIG. 7A has a DNA sequence of

(SEQ ID NO: 33) TACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCGTCTCCCTTATCGTCAATCTTCAAGAGAAAGATTGACGATAAGGGAGATTTTTTTTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCGATAACTCTGTTGTCCTCTTTCAAGAGAAGAGGACAACAGAGTTATCTTTTTTTTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCGCCCCACCGCACGGAGGCCTTTTCAAGAGAAAGGCCTCCGTGCGGTGGGGTTTTTTT.

In the cassette of FIG. 7A each effector complement is operably linkedto a promoter sequence, and the corresponding effector sequence isoperably linked to termination elements or sequences.

In contrast, in the expression cassette of FIG. 7B the first effectorcomplement (effector 1 comp) is operably linked to a promoter and thelast effector (effector 6) is operably linked to a terminator element orsequence, arrows indicate anticipated sites for Dicer processing. Thiscassette is designed to express a single RNA molecule (SEQ ID NO: 44),and has a DNA sequence of

(SEQ ID NO: 34) TACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCGTGCTGTTGACAGTGAGCGTCTCCCTTATCGTCAATCTTCAAGAGAAAGATTGACGATAAGGGAGATGCCTACTGCCTCGGATTCTGCTGTTGACAGTGAGCGGTTGTCCTCTCCCGCAAATACAAGAGATATTTGCGGGAGAGGACAACTGCCTACTGCCTCGGATTCTGCTGTTGACAGTGAGCGCCCCACCGCACGGAGGCCTTCAAGAGAAAGGCCTCCGTGCGGTGGGGTGCTGTTGACAGT GAGCGTTTTTTT.iii) Long Hairpin Expression Cassette

FIG. 8 illustrates the expression cassette, expressed hairpin RNAi agent(SEQ ID NO: 45) and theoretical effector sequence processed by Dicer(assuming 22 nucleotide phasing) based on (in order) SEQ ID NO: 1, SEQID NO:6 and SEQ ID NO:4. In the cassette of FIG. 8, 17 contiguousnucleotides of SEQ ID NO:1 are encoded for and 20 contiguous nucleotidesof SEQ ID NO:6 as per the above examples, but in contrast to thecassettes of FIGS. 7 and 8, only 18 contiguous nucleotides of SEQ IDNO:4 are encoded for. The arrows on the hairpin RNAi agent indicatewhere Dicer is expected to cut to produce the effector sequence shownunderneath.

The expression cassette of FIG. 8 has a DNA sequence of

(SEQ ID NO: 35) TACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCGCTCCCTTATCGTCAATCTTCACCCCACCGCACGGAGGCCTTCTGATGTCCTCTCCCGCAAATACAAGAGATATTTGCGGGAGAGGACAACAGAAGGCCTCCGTGCGGTGGGGTGAAAGATTGACGATAAGGGAGATTTTTTT.

Example 7 Assaying the Effectiveness of ddRNAi Agents In Vitro

To test the efficacy of ddRNAi agents appropriate cells and cell linescan be transfected with RNA agents of the invention and inhibition ofHBV replication assayed

To assay the efficacy of ddRNAi constructs of the invention, plasmidDNAs based on the pGL3 Luciferase Reporter Vector were constructed. Thefirst, “pGL3 multi” contains single copies of sequence in the 3′untranslated regions (UTR) of the firefly luciferase expressing plasmidpGL3, encoding target sites for siRNA or shRNA 3, 9, 12, 13 and 23,based on SEQ ID NOS: 3. 9, 12, 13 and 23 respectively. The second,“pGL3-23” contains a triple repeat of shRNA 23, based on SEQ ID NO:23.The HBV target sites in these plasmids correspond to the sense sequencesof HBV recognised by particular siRNAs or shRNAs and include anadditional 10 nts of HBV sequence upstream and downstream of the targetsites.

The following information relates to experiments conducted using thepGL3-23 construct.

For convenience we chose to undertake initial testing usingdual-luciferase assays with transient assays in HEK293 cells. Cells wereplated in 96-well plates (0.1 ml medium/well) to reach about 50%confluence before transfection. Plasmid DNAs and the chemicalsynthesized siRNAs (or shRNA vectors) were co-transfected into HEK293cells with Lipofectamine™ 2000 using the manufacturer's (Invitrogen)protocol. The test constructs were used together with a constructexpressing Renilla luciferase (pRL-TK, Promega) which acts an internalcontrol for transfection efficiency.

If the chemically synthesized siRNAs, or shRNA expressed from vectorsare effective at silencing the target sequence, luciferase levels intransfected cells will be decreased since the target sequences areoperably linked to the luciferase gene.

Accordingly, various amounts of chemically synthesized siRNAs or shRNAexpression vectors were co-transfected with constant amounts of pGL3-23and control plasmids (see Tables below).

TABLE 5 Testing siRNA23 against pGL3-23 pGL3-23(ng) pRL-TK(ng)siRNA(pmol) siNC (pmol) 20 2 siNC 0 10.0 HBV-siRNA23 0.5 9.5 1.0 9.0 2.57.5 5.0 5.0 10.0 0.0 siRNA-GL3 5.0 5.0

Cells were transfected with indicated amounts of pGL3-23 and control(pRL-TK) plasmids and varying amounts of siRNAs based on SEQ ID NO:23(HBV-siRNA23). siNC, an unrelated siRNA with no known target sequencesin HBV, was used both as a negative control and to adjust totalquantities of siRNAs added to cells to avoid potential artifacts due tounequal transfection. siRNA GL3 was used as a positive control, suchthat it is an siRNA targeted to the luciferase gene.

TABLE 6 Testing shRNA23 against pGL3-23 pGL3-23 (ng) pRL-TK (ng) shRNA(ng) pUC57 (ng) 20 2 pUC57 0 100 HBV shRNA23 2 98 5 95 10 90 20 80 50 50100 0 U6 shRNA-GL3 50 50

Cells were transfected with indicated amounts of pGL3-23 and control(pRL-TK) plasmids and varying amounts of test plasmid for expressingshRNA based on SEQ ID NO:23 (HBV shRNA23). pUC57 was used both as anegative control and to adjust total quantities of plasmid DNAs added tocells to avoid potential artifacts due to unequal transfection. Aplasmid expressing a luciferase shRNA based on GL3 siRNA was used as apositive control.

Samples for testing were transfected into 4 individual wells and fireflyand Renilla Luciferase activities were determined 48 h post-transfectionusing the Dual-Luciferase Assay System (Promega) and a Synergy™ 2Luminescence microplate reader (Biotek) according to the respectivemanufacturer's protocol. The firefly/Renilla activity ratios weredetermined for each well, and the inhibition efficiency of siRNA orshRNAs were calculated by normalizing to respective controls.

Results

The graphs in FIGS. 10A and B show luciferase activities (+/−SD; n=4) incells transfected with varying quantities of chemically synthesisedsiRNA23 (A) or shRNA23 expression constructs (B) targeting pGL3-23 usingthe conditions listed in Tables 5 and 6. Even at the lowestconcentrations, the siRNA and shRNA based on SEQ ID NO:23 aredownregulated relative to the negative control (siNC or shNC—arbitrarilyset at 1). The positive control luciferase siRNA and shRNA alsodownregulated luciferase expression levels from pGL3-23.

Example 8 Assaying the Effectiveness of ddRNAi Agents In Vivo

Mouse models for HBV are available, such as HBV infected NOD/SCID mice(Yang et al. 2002; Ketzinel-Gilad et al. 2006), as well as transgenicmouse lines expressing HBV, either or both of which could be used forthese experiments.

Injection of ddRNAi agents into mouse tail veins will be used to deliverthe agents to the liver. Inhibition of HBV replication can be monitoredat various time points using qRT-PCR assays to determine the levels ofHBV pol mRNA in liver tissues or by quantifying levels of circulatingHBsAg and HBeAg in animals treated with ddRNAi agents of the inventioncompared to appropriate control animals such as mice injected withscrambled sequences or irrelevant DNAs, as described above.

Suitable expression constructs for stable expression of the ddRNAiagents include lentiviral vectors.

Example 9 In Vivo Testing of the Pre-Clinical Candidate in a NormalMouse Model

One delivery system envisaged in vivo testing of the pre-clinicalcandidate in a normal mouse model is AAV. For AAV, the ddRNAi constructswill be packaged in vitro using strategies well known to those familiarwith the art and injected intravenously into HBV infected NOD/SCID miceand/or HBV transgenic mice. Inhibition of HBV replication will bemonitored as described above.

It will be understood that the invention disclosed and defined in thisspecification extends to all alternative combinations of two or more ofthe individual features mentioned or evident from the text or drawings.All of these different combinations constitute various alternativeaspects of the invention.

What is claimed is:
 1. A DNA-directed RNA interference (ddRNAi) agentfor inhibiting expression of one or more target sequences in one or moreHepatitis B virus (HBV) genes, the ddRNAi agent comprising: a firsteffector sequence of at least 17 nucleotides in length; a secondeffector sequence of at least 17 nucleotides in length; a secondeffector complement sequence; and a first effector complement sequence;wherein each effector sequence is substantially complementary to thepredicted transcript of a target sequence.
 2. A ddRNAi agent accordingto claim 1, comprising a third effector sequence of at least 17nucleotides in length and a third effector complement sequence.
 3. AddRNAi agent according to claim 1 comprising, in a 5′ to 3′ direction: afirst effector sequence of at least 17 nucleotides in length; a firsteffector complement sequence; a second effector sequence of at least 17nucleotides in length; and a second effector complement sequence.
 4. AddRNAi agent according to claim 2 comprising in a 5′ to 3′ direction: afirst effector sequence of at least 17 nucleotides in length; a firsteffector complement sequence; a second effector sequence of at least 17nucleotides in length; a second effector complement sequence; a thirdeffector sequence of at least 17 nucleotides in length; and a thirdeffector complement sequence.
 5. A ddRNAi agent according to claim 1,wherein each effector sequence is selected from the group consisting ofany 10 or more contiguous nucleotides within a sequence from any one ofSEQ ID NOS: 1 to
 27. 6. A ddRNAi agent according to claim 5, whereineach effector sequence is selected from SEQ ID NOS: 3, 9, 12, 13 and 23.7. A ddRNAi expression cassette for expressing a ddRNAi agent accordingto claim 1, the expression cassette comprising: one or more promotersequences; one or more DNA sequences that encode for one or moreeffector sequences; one or more DNA sequences that encode for one ormore effector complement sequences; and one or more terminatorsequences; and optionally: one or more DNA sequences that encode forloop sequences; and/or one or more enhancer sequences.
 8. A ddRNAiexpression cassette according to claim 7, wherein the one or more DNAsequences encode for one or more effector sequences selected from thegroup consisting of any 10 or more contiguous nucleotides within asequence from any one of SEQ ID NOS: 1 to
 27. 9. A ddRNAi expressionconstruct comprising a ddRNAi expression cassette according to claim 7.10. A method of treating acute or chronic HBV infection in a subjectcomprising administering a therapeutically effective amount of a ddRNAiagent according to claim
 1. 11. A method of reducing HBV viral load in asubject comprising administering a therapeutically effective amount of addRNAi agent according to claim
 1. 12. A method of reducing the severityof symptoms associated with HBV infection in a subject comprisingadministering a therapeutically effective amount of a ddRNAi agentaccording to claim
 1. 13. A method of reducing the infectivity of HBVcomprising administering a therapeutically effective amount of a ddRNAiagent according to claim
 1. 14. A method according to claim 10, whereinthe ddRNAi agent inhibits expression of at least the HBV polymerasegene.
 15. A pharmaceutical composition comprising a ddRNAi agentaccording to claim 1 and a pharmaceutically acceptable carrier ordiluent.
 16. A method of treating acute or chronic HBV infection in asubject comprising administering a therapeutically effective amount of addRNAi expression construct of claim
 9. 17. A method of reducing HBVviral load in a subject comprising administering a therapeuticallyeffective amount of a ddRNAi expression construct of claim
 9. 18. Amethod of reducing the severity of symptoms associated with HBVinfection in a subject comprising administering a therapeuticallyeffective amount of a ddRNAi expression construct of claim
 9. 19. Amethod of reducing the infectivity of HBV comprising administering atherapeutically effective amount of a ddRNAi expression construct ofclaim
 9. 20. A pharmaceutical composition comprising a ddRNAi expressionconstruct according to claim 9 and a pharmaceutically acceptable carrieror diluent.